& 


.    >O 


UNIVERSITY  OF  ILLINOIS 

Agricultural  Experiment  Station 


BULLETIN  No.  202 


IS  SYMBIOSIS  POSSIBLE  BETWEEN  LEGUME 
BACTERIA  AND  NON-LEGUME  PLANTS? 


BY  THOMAS  J.  BURRILL  AND  ROY  HANSEN 


URBANA,  ILLINOIS,  JULY,  1917 


CONTENTS  OF  BULLETIN  No.  202 

PAGE 

INTRODUCTION , 115 

PAET  I.     THE  OEGANISM  '. 116 

Isolation  and  Cultivation 116 

Morphology   118 

Cultural  Characteristics 123 

PAET  II.     CEOSS-INOCULATIONS:     VABIETIES    OP    NODULE    BAC- 

TEEIA -.125 

Cross-Inoculation  Investigations   125 

Grouping  by  Serologieal  Tests  and  by  Cultural  Differences 137 

PAET  III.     HISTOLOGY  OF  THE  NODULES  OF  THE  LEGUMINOSAE . .  141 

Origin  of  the  Nodule 142 

Structure   of  the  Nodule 142 

PAET  IV.     NON-LEGUMES  SAID  TO  BE  CONCEENED  IN  THE  FIXA- 
TION OF  ATMOSPHEEIC  NITEOGEN 145 

Historical 145 

Ceanothus  americanus 145 

Cycas  revoluta    148 

Alnus,  Elaeagnus,  and  Myrica 149 

Conclusions 150 

PAET   V.     ATTEMPTS  TO  DEVELOP   A  SYMBIOSIS  BETWEEN  LE- 
GUME BACTEEIA  AND  NON-LEGUME  PLANTS 151 

Evidence  of  Constancy  or  Change  in  the  Organism 152 

•    Experiments  Attempting  the  Infection  of  Non-Legume  Plants  with  Ps. 

radicicola 155 

SUMMAE.Y 160 

PAET  VI.     BIBLIOGEAPHIES 161 

Symbiotic  Nitrogen  Fixation  by  Legumes 161 

Non-Legume  Boot  Nodules 179 


ILLUSTRATIONS 

PLATE 

I.  Fig.  1. — Ash-agar  plate  from  bean.  Fig.  2. — Ash-agar  plate  from  per- 
ennial pea 

II.  Fig.  1. — Ash-agar  plate  from  pea.  Fig.  2. — Ash-agar  plate  from  dyer's 
greenweed 

III.  Fig.  1. — Bacteroids  from  a  very  young  nodule  of  pea.     Fig.  2. — Bac- 

teroids  from  young  growing  nodule  of  hairy  vetch.     Fig.  3. — Bac- 
teroids from  an  older  nodule  of  hairy  vetch 

IV.  Pseudomonas  radicicola,  showing  polar  flagellum :    organisms  from  cow- 

pea,  partridge  pea,  acacia,  tick  trefoil,  and  Japan  clover 

V.  Pseudomonas  radicicola,  showing  polar  flagellum :  organisms  from  velvet 
bean,  peanut,  wild  indigo,  hog  peanut,  soybean;  also  B.  subtilis  in- 
troduced for  comparison 

VI.     Seedlings  of  partridge  pea  inoculated  with  bacteria  from  cowpea 
VII.     Seedlings  of  cowpea  inoculated  with  bacteria  from  partridge  pea 
VIII.     Seedlings  of  cowpea  inoculated  with  bacteria  from  six  species  of  Acacia 

IX.  Seedlings  of  cowpea  inoculated  with  organisms  from  partridge  pea,  tick 
trefoil,  dyer's  greenweed,  Japan  clov.er,  velvet  bean,  cowpea,  acacia, 
peanut,  wild  indigo 

X.  Seedlings  of  alfalfa  grown  after  Garman  's  method,  showing  inoculation 
by  several  cultures 

XL  Fig.  1. — Longitudinal  section  of  a  nodule  of  red  clover.  Fig.  2. — Cross- 
section  of  a  similar  nodule  of  red  clover 

XII.  Fig.  1. — Cross-section  thru  the  meristem  region  of  a  nodule  of  hairy 
vetch.  Fig.  2. — Cross-section  thru  the  same  nodule  some  distance  back 
from  the  meristem.  Fig.  3. — Infection  threads  in  the-  cortex  cells  of 
a  nodule  of  red  clover 

XIII.  Fig.  1. — Young  infected  cells  of  a  nodule  of  hairy  vetch.    Fig.  2. — Bac- 

teroidal  cells  of  red  clover  in  a  well  advanced  but  growing  nodule 

XIV.  Eoot  nodules  of  Ceanothus  americanus 

XV.  Fig.  1. — Longitudinal  section  of  a  Ceanothus  americanus  nodule.  Fig. 
2. — Cross-section  thru  a  similar  nodule  of  Ceanothus  americanus 

XVI.  Fig.  1. — Parasitized  cells  of  a  Ceanothus  americanus  nodule.  Figs.  2 
and  3. — Same  more  highly  magnified 

XVII.     Experiment   VIII :      Morning-glory  plants   inoculated  with   sweet-clover 
bacteria 


FOREWORD 

This  bulletin  reports  the  last  work  of  Thomas  Jonathan  Bur  rill, 
who  in  1880,  thru  studies  of  pear  blight,  first  experimentally  proved 
the  fact  that  plant  diseases  are  sometimes  caused  by  bacterial  invasion. 

Symbiotic  relationships  early  attracted  the  attention  of  Dr.  Bur- 
rill,  especially  the  relationship  existing  between  certain  nitrogen- 
gathering  bacteria  and  legumes.  In  those  days  every  discovery  gave 
rise  to  new  and  fundamental  questions,  and  the  query  whether  such 
relation  is  necessarily  confined  to  legumes  was  always  in  his  mind,  and 
was  put  aside  only  by  the  urgency  of  pressing  duties. 

When  after  retirement  from  active  service,  opportunity  came  to 
Dr.  Burrill  for  following  his  inclination,  his  attention  at  once  reverted 
to  the  old-time  problem,  and  he  fitted  up  a  laboratory  and  employed 
an  assistant  for  its  study.  Here  he  devoted  the  last  three  years  of  his 
life,  and  here  the  call  came  suddenly  on  April  14,  1916,  forty-eight 
years  to  a  month  after  his  coming  to  the  University  of  Illinois. 

Especial  credit  is  due  the  junior  author  for  his  faithful  and  hope- 
fully successful  attempt  at  accurately  reporting  the  work  as  planned 
by  his  chief,  and  so  far  as  is  humanly  possible  correctly  interpreting 
his  ideas  and  convictions. 

In  this  difficult  task  Mr.  Hansen  has  been  aided  by  Dr.  A.  L. 
Whiting  with  some  special  knowledge  of  the  technical  material  in- 
volved, and  by  Professor  C.  F.  Hottes,  for  a  quarter  of  a  century  Dr. 
Burrill 's  close  associate,  who  has  read  the  manuscript  with  the  view  of 
insuring  that  so  far  as  possible  the  spirit  and  thought  of  the  pioneer 
investigator  is  expressed. 

E.  DAVENPORT 

Director 


IS  SYMBIOSIS  POSSIBLE  BETWEEN  LEGUME 
BACTERIA  AND  NON-LEGUME  PLANTS? 

By  THOMAS  J.  BURRILL,  PROFESSOR  OF  BOTANY,  EMERITUS,  AND 
EOY  HANSEN,  ASSISTANT  IN  NITROGEN-FIXATION  RESEARCH 

INTRODUCTION 

The  work  reported  in  this  bulletin  deals  with  an  attempt  to 
develop  a  symbiosis  between  legume  bacteria  and  non-legume  plants 
similar  to  that  which  exists  between  legume  bacteria  (Pseudomonas 
radicicola)  and  legume  plants. 

Since  the  demonstration,  in  1886,  by  Hellriegel  and  Wilfarth  of  the 
symbiotic  fixation  of  atmospheric  nitrogen  by  legume  plants  and  cer- 
tain microorganisms,  no  crop  rotation  has  been  considered  rational  that 
does  not  include  a  liberal  use  of  legumes.  The  importance  of  this 
discovery  to  agriculture  is  generally  appreciated.  That  it  is  applicable 
thruout  the  world  makes  it  of  especial  value  to  mankind. 

The  benefit  that  would  result  could  other  ordinary  farm  crops  be 
enabled  to  utilize  atmospheric  nitrogen  would  be  inestimable;  hence 
the  importance  of  any  success  in  this  direction.  In  attempting  to 
study  this  question  it  was  fully  realized  that  success  might  not  be 
attained,  but  that  it  was  in  the  realm  of  possibilities.  It  was  nearly 
a  quarter  of  a  century  ago  that  the  first  work  was  done  under  the 
direction  of  the  senior  author.  Since  that  time  a  few  attempts  have 
been  made  by  other  workers  to  grow  legume  bacteria  on  mustard  and 
grasses,  but  with  negative  results. 

In  returning  to  this  problem,  the  authors  found  it  necessary  at 
first  to  spend  considerable  time  in  acquiring  an  intimate  acquaintance 
with  the  organism  concerned,  especially  in  regard  to  its  cultivation 
and  identification.  Attention  was  given  to  the  special  adaptations,  or 
varieties,  of  the  symbiotic  bacteria  in  order  to  learn,  first,  whether 
these  adaptations  were  constant  or  subject  to  change ;  and  second,  what 
factors  were  responsible  for  their  existence.  Histological  studies  of 
the  nodule  were  undertaken  with  the  view  of  learning  something  of  the 
relations  existing  between  the  two  symbionts.  The  nodules  of  certain 
non-legume  plants  (Ceanothus,  Cycas,  Elaeagnus,  etc.),  said  to  be 
concerned  in  the  fixation  of  atmospheric  nitrogen,  were  given  some 
attention  in  the  hope  that  perhaps  here  lay  a  start.  Cross-inoculations 
of  importance  and  interest  were  found  and  are  reported  as  a  part  of 
this  contribution.  Some  preliminary  trials  were  made  attempting  the 
inoculation  of  non-legume  plants  with  the  legume  organism. 

115 


116 


BULLETIN  No.  202 


[July, 


Part  I.— THE  ORGANISM 
ISOLATION  AND  CULTIVATION 

Media. — Pseudomonas  radicicola  was  cultivated  on  many  kinds  of 
media  differing  widely  in  composition,  and  it  was  found  that  it  would 
thrive  on  most  of  them.  For  plating  out,  Harrison  and  Barlow's 
wood-ash  agar  was  usually  used,  as  it  gave  more  uniform  results. 
Many  media  were  unsuitable  for  plating,  yet  permitted  growth  upon 
agar  slants. 

A  list  of  media  employed  in  these  experiments  for  cultivating 
Ps.  radicicola,  together  with  the  composition  and  reaction  of  each,  is 
given  in  Table  1. 


TABLE  1. — COMPOSITION  AND  KEACTION  OF  MEDIA  USED  IN 
CULTIVATING  Pseudomonas  radicicola 


Labora- 
tory No. 

Medium 

Composition 

Reaction* 

100 

Wood  ash 
(Harrison  and 
Barlow) 

Wood-ash     extract     (15 
gins,    ashes    to    1    liter 
tap  water)                         1000     cc. 
Saccharose                               10     gms. 
Monopotassium  phosphate       3     gms. 

Not  changed; 
usually   -(-7°   to 
-4-10°  to  phenol  - 
phthalein 

101 

Synthetic 
(Fred) 

Distilled  water                    1000     cc. 
Dextrose                                  20     gms. 
Monopotassium  phosphate       1     gm. 
Magnesium  sulfate                     .1  gm. 
Sodium  chlorid                   Trace 
Ferrous  sulfate 
Manganous  sulfate 
Calcium  chlorid                       " 

Titrate  to  +10° 

102 

Mannite 
(Ashby) 

Distilled  water                    1000     ce. 
Mannite                                   20     gms. 
Dipotassium  phosphate             .2  gm. 
Magnesium  sulfate                    .2  gm. 
Sodium  chlorid                            .2  gm. 
Calcium  sulfate                          .1  gm. 
Calcium  carbonate                   5     gms. 

Not  changed 

103 

Synthetic 
(Spratt) 

Distilled  water                      100     cc. 
Cane  sugar      '                         1     gm. 
Dipotassium  phosphate              .5  gm. 
Magnesium  sulfate                     .02  gm. 
Calcium  carbonate                     .1  gm. 

Titrate  to  +10° 

104 

Asparaginate 
(Conn) 

Distilled  water                    1000     cc. 
Sodium  asparaginate               1     gm. 
Dextrose                                    1     gm. 
Magnesium  sulfate                    .2  gm. 
Ammonium  phosphate"           1.5  gms. 
Calcium  chlorid                            .1  gm. 
Potassium  chlorid                      .1  gm. 
Ferric  chlorid                       Trace 

Not  changed; 
usually  +6° 
to  +8° 

•Fuller's  scale  in  all  cases. 

bUsed  in  place  of  mono-ammonium  phosphate. 


1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        117 

TABLE  1. — Continued 


Labora- 
tory No. 

Medium 

Composition 

Eeaction 

105 

Beef  broth 

Tap  water                         1000    cc. 
Witte's  peptone                     10     gms. 
Beef  extract  (Liebig's)          5     gms. 

Titrate  to  +10° 

106 

Legume  extract, 
using  bean 
plant 

Extract   of    bean   plant 
(Heat    100    gms.    roots 
and  stems  in  1  liter  tap 
water  y2  hour  at  60°  C.)  1000     cc. 
Cane  sugar                              20     gms. 

Titrate  to  +10° 

107 

Bean-extract 
peptone 

Same  as  106,  plus  1  percent  peptone 
(Witte's) 

Titrate  to  +10° 

108 

Legume  extract, 
using  sweet 
clover 

Sweet-clover  extract        1000     ce. 
Cane  sugar                              20     gms. 

Titrate  to  +10° 

109 

Sweet-clover  - 
extract  pep- 
tone 

Same  as  108,  plus  1  percent  peptone 

Titrate  to  +10° 

110 

Tomato 
infusion 

Tomato     extract      (100 
gms.  plant  substance  to 
1  liter  water)                      1000  cc. 
Cane  sugar                                20  gms. 

Titrate  to  -flO° 

111 

Tomato-infu- 
sion peptone 

Same  as  110,  plus  1  percent  peptone 

Titrate  to  -f  10° 

200 

Wood-ash  agar 

Same  as  100,  plus  1  percent  agar 

Not  changed 

201 

Synthetic  agar 
(Fred) 

Same  as  101,  plus  1  percent  agar 

Titrate  to  +10° 

202 

Mannite  agar 
(Ashby) 

Same  as  102,  plus  1  percent  agar 

Not  changed 

203 

Synthetic  agar 
(Spratt) 

Same  as  103,  plus  1  percent  agar 

Titrate  to  +10° 

204 

Asparaginate 
agar 

Same  as  104,  plus  1  percent  agar 

Not  changed 

205 

Beef  -broth  agar 

Same  at  105,  plus  1  percent  agar 

Titrate  to  +10° 

206 

Bean-extract 
agar 

Same  as  106,  plus  1  percent  agar 

Titrate  to  +10° 

207 

Bean-extract- 
peptone  agar 

Same  as  107,  plus  1  percent  agar 

Titrate  to  +10° 

208 

Sweet-clover-ex- 
tract agar 

Same  as  108,  plus  1  percent  agar 

Titrate  to  +10° 

209 

Sweet-clover-ex- 
tract-peptone 
agar 

Same  as  109,  plus  1  percent  agar 

Titrate  to  +10° 

210 

Tomato-extract 
agar 

Same  as  110,  plus  1  percent  agar 

Titrate  to  +10° 

211 

Tomato-extract- 
peptone  agar 

Same  as  111,  plus  1  percent  agar 

Titrate  to  4-10° 

300 

Wood-ash 
gelatin 

Same  as  100,  plus  12  percent  gelatin 

Not  changed 

305 

Beef-broth 
gelatin 

Same  as  105,  plus  12  percent  gelatin 

Titrate  to  -f  10° 

420 

Potato  slant 

421 

Tomato-stem 
slant 

Fresh  young  tomato  stems  in  distilled 
water 

118  BULLETIN  No.  202 

Isolation.— In  isolating  the  organism  the  following  method 
adapted  from  that  of  Harrison  and  Barlow32"  was  used:  Where 
choice  is  possible  select  a  medium  sized  nodule  appearing  young  and 
sound.  In  cutting  it  off  leave  two  or  three  millimeters  of  the  root  on 
both  sides  of  the  nodule  to  permit  handling  it  with  forceps.  Wash 
carefully,  rinse  in  distilled  water,  and  drop  into  a  sterilizing  fluid 
made  as  follows : 

Distilled  water 500  cc. 

Bichlorid  of  mercury 1  gin. 

Hydroclorie  acid  (C.  P.) 2.5  cc. 

Shake  the  nodule  violently  in  this  solution  for  one  or  two  minutes, 
after  which  wash  it  three  times  with  sterile  distilled  water.  Then 
cover  with  about  1  cc.  of  sterile  distilled  water  and  crush  with  a 
heavy  glass  rod,  previously  flamed  and  cooled.  Pour  two  or  three 
drops  of  the  cloudy  suspension  into  a  test  tube  of  ash  agarb  at  45°  C. 
Inoculate  a  second  tube  of  the  agar  with  five  loops  from  the  first,  and 
pour  plates.  When  a  large  nodule  is  used,  inoculate  the  first  tube 
with  five  loops  of  the  suspension,  and  inoculate  the  second  tube  with 
five  loops  from  the  first,  and  pour  plates:  Only  two  plates  are  poured ; 
a  third  was  found  unnecessary.  Incubate  plates  at  from  20°  to  25°  C. 
Replating  is  usually  unnecessary,  altho  it  is  a  safe  practice  with  ques- 
tionable plates.  If  the  sterilization  and  washing  are  carefully  done, 
foreign  organisms  seldom  appear. 

Cultivation. — For  keeping  stock  cultures  ash  agar  was  used. 
Transfers  were  made  once  a  month,  tho  cultures  may  easily  be  kept 
six  weeks  or  two  months  between  transfers.  Plate  colonies  should  be 
large  enough  for  transfer  in  six  to  fourteen  days,  depending  upon 
the  host  plant  used  and  other  conditions. 

MORPHOLOGY 

Agar  Colonies. — In  general  the  colonies  appearing  on  agar  plates 
may  be  divided  into  two  types,  buried  and  surface  colonies. 

Buried  colonies  are  small  and  submerged,  most  frequently  lens, 
or  spindle  shaped,  with  smooth  and  even  edges.  They  are  quite 
opaque,  granular  in  structure,  and  in  color  are  cream  to  a  chalk  white. 
They  increase  slowly  in  size,  eventually  appearing  on  the  surface  of 
the  agar  as  surface  colonies,  when  the  growth  becomes  rapid.  The 


•Superior  figures  are  used  to  indicate  the  literature  citations  having  special 
reference  to  this  work  which  are  given  in  the  bibliographies. 

"Obtain  ashes  from  thoroly  burned  hard  wood  and  run  thru  a  fine  sieve.  (Little 
difference  was  found  in  different  lots  of  ashes.)  "Use  15  grams  to  one  liter  of  tap 
water  and  bring  to  a  boil  over  a  free  flame,  stirring  at  intervals.  Allow  solution  to 
stand  from  five  to  ten  minutes,  then  filter  thru  a  double  filter.  To  one  liter  of  ash 
extract  add  10  grams  of  saccharose,  3  grams  of  KH2PO<,  and  10  grams  of  agar. 
Autoclave  for  fifteen  minutes,  filter  thru  absorbent  cotton,  and  proceed  as  with 
other  media.  The  reaction  usually  was  -f7°  to  +10°  (Fuller's  scale)  to  phenol- 
phthalein,  and  was  never  changed. 


1017]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  &ACTERIA  AND  NON-LEGUMES        119 

lens  colonies,  however,  remain  visible  for  many  days  in  the  center  of 
the  new  growth. 

Surface  colonies  originate  at  or  near  the  surface  of  the  agar  or 
develop  from  buried  colonies.  They  are  drop-form,  watery,  mucilagi- 
nous (in  appearance,  tho  not  always  to  the  touch),  gray-white  to 
pearly  white  in  color,  glistening,  and  semitranslucent  to  opaque.  The 
edges  are  smooth  and  even.  Under  the  low  power  the  interior  is  granu- 
lar. They  frequently  attain  considerable  size,  a  centimeter  or  more  in 
diameter. 

Plates  made  direct  from  the  nodule  lack  uniformity  to  a  marked 
degree.  The  undiluted  plate  (first  plate)  begins  to  show  a  few  colonies 
in  two  to  four  days.  These  colonies  become  extremely  large  in  a  very 
short  time,  their  rapid  growth  being  due  to  small  pieces  of  nodule 
tissue  or  to  clumps  of  bacteria  carried  over  into  the  agar  (see  Plate  I). 
In  five  or  six  days  numerous  colonies  begin  to  make  their  appearance, 
most  of  them  as  submerged  colonies,  which  later  grow  to  the  surface. 

The  dilution-plate  (second-plate)  colonies  are  always  extremely 
slow  in  growth.  Generally  colonies  are  large  enough  for  transfer  in 
six  to  fourteen  days,  tho  plates  should  not  be  discarded  for  two  or  even 
three  weeks. 

The  rate  of  growth  of  colonies  also  varies  with  the  organisms  of 
different  nodules  (see  Plate  II).  Among  the  fast  growers  are  the 
organisms  from  the  pea  (Pisum),  vetch  (Vicia),  lentil  (Lens),  sweet 
pea  (Lafhyrus),  bean  (Phaseolus) ,  lupine  (Lupinus),  wild  bean 
(Strophostyles) ,  clover  (Trifolium),  sweet  clover  (Melilotus),  alfalfa 
(Medicago),  and  fenugreek  (Trigonella).  The  organisms  appreciably 
slower  in  growth  are  those  from  the  cowpea  (Vigna),  Japan  clover 
(Lespedeza),  tick  trefoil  (Desmodium),  acacia  (Acacia), partridge  pea 
(Cassia),  false  indigo  (Baptisia),  dyer's  greenweed  (Genista),  peanut 
(Arachis),  soybean  (Glycine),  and  hog  peanut  (Amphicarpa) . 

The  Bacteria. — The  life  cycle  of  Pseudomonas  radicicola  from 
the  soil  thru  the  nodule  and  back  to  the  soil  is  clouded  in  doubt 
because  of  the  extreme  variability  of  the  organism  under  apparently 
the  same  conditions.  While  it  has  been  isolated  from  soil  (see  Lip- 
man41  42),  there  is  no  clue  to  the  form  in  which  it  existed  in  the  soil. 

Observation  of  cowpea  nodules  showed  that  in  the  very  young 
nodules  there  is  considerable  variation  in  size  and  shape  of  the  organ- 
isms. Many  of  the  small,  oval  forms,  the  swarmers  described  by 
Beyerinck,9  are  found.  These  forms  and  the  normal  rods  predomi- 
nate. Large  club-shaped  bacteroids  are  frequent;  the  characteristic 
branched  forms  are  not  so  numerous.  The  bacteroids  are  best  demon- 
strated when  the  young  nodule  is  just  beginning  to  show  a  reddish 
interior.  At  this  stage  they  are  extremely  large  and  contain  the  maxi- 
mum staining  substance  (see  Plate  III).  The  characteristic  X  and 
Y  forms  occur  in  great  numbers ;  they  show  considerable  vacuolation 
and  unevenness  in  staining,  especially  when  stained  with  carbol- 
fuchsin. 


120 


BULLETIN  No.  202 


PLATE  I 

Fig.  1. — Ash-agar  plate  from  bean  (Pkaseolus  vulgaris},  showing  giant  col- 
onies in  a  thickly  seeded  plate 

Fig.  2. — Ash-agar  plate  from  perennial  pea  (Lathyrus  latifolius) ;  the  clear 
spaces  are  due  to  sterilizing  fluid  carried  over  with  pieces  of 
nodule  tissue 


1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        121 

In  the  old,  decomposing  nodule  the  bacteroids  are  extremely 
vacuolated  and  ghost-like,  showing  small,  oval,  deep-staining  bodies 
within.  The  inference  is  that  these  bodies  are  motile  swarmers,  which 
later  free  themselves  from  the  ghost-like  capsules,  rather  than  bud 
off,  as  has  been  described  by  some  writers.  Frequently  the  swollen 
rods  have  a  beaded  appearance  with  unstained  bands  or  areas.  A 
few  motile  rods  may  sometimes  be  seen  in  hanging  drops  in  this  stage, 
and  sometimes  a  bacteroid  is  seen  to  oscillate  as  tho  swung  about  by 
some  propelling  force  in  one  end.  Division  of  the  bacteroids  into 
bacilli,  as  represented  by  Dawson,23  may  also  occur. 

When  first  plated  out,  the  young  colonies  consist  of  small  rods 
which  show  considerable  variation  in  length.  No  bacteroids  are  pres- 
ent, tho  the  rods  are  sometimes  slightly  club-shaped  and  sometimes 
show  vacuolation.  However,  they  never  attain  the  size  of  bacteroids. 
With  frequent  transfers  the  rods  become  quite  uniform  in  size  and 
slain  deeply  and  evenly,  especially  with  aniline-gentian-violet. 

In  very  old  cultures  (three  months  on  ash  agar,  without  transfer) 
the  small,  oval  swarmers  and  the  normal  rods  predominate,  tho  a  few 
club-shaped  and  a  few  branched  bacteroids  are  found.  The  bacteroids 
produced  upon  artificial  media*  are  never  so  large  nor  so  numerous  as 
those  seen  in  mounts  direct  from  a  young  nodule. 

Staining. — The  organisms  do  not  stain  well  with  ordinary  aniline 
stains.  Carbol-fuchsin  and  aniline-gentian-violet  (used  steaming)  are 
the  most  satisfactory  stains.  Tho  carbol-fuchsin  was  preferred,  aniline- 
gentian-violet  stains  were  always  used  as  checks,  because  the  former 
slain  accents  the  vacuolated  appearance,  particularly  in  bacteroids. 
Carbol-fuchsin  is  especially  useful  in  staining  bacteroids  direct  from 
the  nodule  and  also  old  agar  cultures.  Kiskalt's  amyl-gram  stain, 
described  by  Harrison  and  Barlow,32  is  useful  since  the  amyl  alcohol 
clears  up  the  field,  leaving  the  bacteria  stained,  tho  not  so  intensely. 
This  stain,  however,  should  not  be  considered  a  means  of  identifying 
Ps.  radicicola. 

Bacteroids. — While  Ps.  radicicola  produces  no  spores,  it  produces 
bacteroids  which  are  very  evidently  more  resistant  than  the  normal 
rods.  Unfavorable  conditions,  such  as  unsuitable  media,  infrequent 
transfer,  or  addition  of  caffein  to  the  medium,  cause  their  appearance. 
This  is  in  accord  with  what  takes  place  in  the  nodule.  In  the  growing 
nodule,  when  development  is  most  rapid,  the  bacteroids  are  at  their 
maximum ;  they  enable  the  organisms  to  multiply  rapidly  in  spite  of 
the  resistance  offered  by  the  plant  cells.  Transferred  to  favorable 
media  from  this  stage  the  normal  uniform  bacilli  are  produced.  The 
bacteroid,  then,  must  be  regarded  as  a  normal  and  a  very  necessary 


•From  the  writers'  observations  this  is  equally  true  of  the  bacteroids  produced 
by  adding  caffein  to  a  legume-extract-agar  medium,  according  to  the  method  of 
Z'ipfel37  and  Fred." 


122 


BULLETIN  No.  202 


[July, 


stage  in  the  life  of  the  organism.  Its  significance  in  the  actual  fixation 
of  nitrogen,  however,  is  pure  speculation. 

Motility. — The  motility  of  the  organism  is  best  seen  in  young  agar- 
slant  cultures,  twenty-four  to  forty-eight  hours  old.  The  bacteria 
dart  about  with  amazing  rapidity,  now  tumbling  end  over  end,  now 
spinning  violently  on  the  shorter  axis,  and  then  sweeping  across  the 
field  in  a  darting,  jerky  course. 

Flagella. — Owing  to  the  gum  or  slime  produced  by  the  organism, 
the  demonstration  of  flagella  is  especially  difficult.  The  lack  of  agree- 
ment among  investigators  as  to  the  number  is  shown  in  Table  2.  The 
organisms  reported  by  these  investigators  were  all  the  most  abundant 
producers  of  gum. 

TABLE  2. — RESULTS  OF  PREVIOUS  WORK  UPON  FLAGELLA  STAINS 


Investigator 

Source  of 
organism 

Flagella 

Remarks 

Beyerinck 
1888 

One  polar  flagellum 

Inferred  during  slow 
motility  and  not  seen 

Smith,  R.  G.20 
1899 

Exceedingly  thin, 
single,  terminal  flagel- 
lum about  2  microns 
long  and  bearing  up- 
on the  distal  end  a 
tuft,  like  the  lash  of 
a  whip 

One  photomicrograph 

Harrison 
and  Barlow 
1907 

Hairy  vetch 

(Vicia  villosa}          . 
Perennial  pea 
(Lathyrus  sativus*) 
Bean 
(Phaseolus  vulgaris)  . 

Single  polar  flagellum 

Several  figures;  mu- 
cilage, or  negative 
method,  by  which 
slime  is  stained  leav- 
i  n  g  flagella  u  n- 
stained;  discredited 
by  Kellerman 

De  Eossi30 
1907 

Broad  bean 
(Vicia  faba) 

Bacillus 

No  figures;  describes 
white,  non-liquefy- 
ing, non-infectuous 
intruder  which  has  a 
polar  flagellum 

De  Rossi33 
1909 

White  clover 
(Trifolium  repens} 
and  other  clovers 

8  to  10  flagella;  peri- 
triehic 

One  photomicrograph 
of  Trifolium  repens; 
very  good 

Kellerman 
1912 

Garden  pea 
(Pisum  sativum) 
Lima  bean 
(Phaseolus  lunatus) 
Alfalfa 
(Medicago  sativa) 

Flagella  fairly  nu- 
merous; peritrichic 

Three  photomicro- 
graphs, none  of  which 
is  convincing 

Zipfel 
1912 

Numerous  flagella; 
peritrichie 

No  figures 

Prucha43 
1915 

Canada  field  pea 

(Pisum  sativum 
arvense) 

Peritrichic  ;  largest 
number  observed  was 
six,  but  there  may  be 
more 

No  figures 

1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        123 

Organisms  from  red  clover  (Trifolium  pratense),  broad  bean 
(Vicia  faba),  hairy  vetch  (Vicia  vttlosa),  common  bean  (PJiaseolus 
vulgaris),  sweet  clover  (Melilotus  alba),  alfalfa  (Medicago  sativa), 
field  pea  (Pisum  arvense),  and  sweet  pea  (Lafhyrus  odoratus) 
were  stained  for  flagella,  using  several  methods,  but  the  gum  stained 
so  heavily  that  none  could  be  seen.  The  production  of  gum  by  the 
organism,  as  will  be  shown  later,  depends  more  upon  the  plant  species 
from  which  it  is  isolated  than  upon  the  culture  medium.  Attention 
was  then  turned  to  the  organisms  making  less  vigorous  growth,  which 
produce  less  gum.  Successful  stains  were  made  of  the  organisms 
from  cowpea  (Vigna  sinensis),  tick  trefoil  (Desmodium  canescens), 
dyer's  greenweed  (Genista  tinctoria),  velvet  bean  (Mucuna  utilis), 
peanut  (Arachis  liypogoea),  wild  indigo  (Baptisia  tinctoria),  Japan 
clover  (Lespedeza  striata),  acacia  (Acacia  floribunda),  partridge 
pea  (Cassia  chamaecrista)  *  soybean  (iGlycine  tiispida),  and  hog  pea- 
nut (Amphicarpa  monoica). 

Loeffler's  method  of  staining  was  used.  The  mordant"  was  made 
up  as  follows : 

Solution  of  tannin  (20  percent  in  water) 10  parts 

Saturated    (cold)    aqueous  solution  of  ferrous 

sulf ate 5  parts 

Saturated  alcoholic  solution  of  basic  f uchsin ...   1  part 

Transfer  the  organisms  successively  several  times  upon  ash  agar 
to  hasten  the  growth.  With  a  platinum  needle  transfer  some  of  the 
organisms  from  the  edge  of  a  transfer  two  or  three  days  old  to  a  small 
drop  "of  sterile  water  upon  a  clean  cover  slip.  Spread  slowly  and  care- 
fully (only  a  few  strokes  are  necessary),  and  allow  to  dry.  Cover 
well  with  a  mordant,  bring  to  a  steam,  and  allow  to  stand  about  one 
minute.  Wash  carefully  with  distilled  water  and  apply  carbol-fuchsin, 
bring  to  a  steam,  and  again  let  stand  one  minute. 

In  examining  the  slide,  look  especially  near  the  edges  of  the  smear 
and  close  to  the  "drifts"  of  bacteria.  Slime  and  stain  deposits  fre- 
quently interfere,  tho  not  seriously.  The  organism  has  a  single  polar 
flagellum  (see  Plates  IV  and  V).  It  was  noted  that  the  flagellum  is 
rarely  attached  at  the  end,  but  rather  at  a  corner. 

CULTURAL  CHARACTERISTICS 

Ps.  radicicola  will  grow  between  0°  and  50°  C.  The  optimum 
temperature  is  25°  to  28°  C.,  tho  it  will  grow  well  at  room  tem- 
perature, or  20°  to  25°C.  The  organism  is  aerobic.  The  diffused  light 


•The  organisms  of  these  nine  plants  comprize  a  single  group,  i.  e.,  they  are 
indentical,  as  will  be  shown  later.  Isolations,  however,  were  made  from  the  host 
plants  as  named.  Actually,  then,  but  three  distinct  varieties  were  stained — Vigna, 
Glycine,  and  AmpJiicarpa. 

"Filter  the  ingredients  separately  and  mix  in  the  order  given.  Filter  direct' 
upon  the  cover  slip.  The  mordant  is  best  used  fresh. 


124  .  BULLETIN  No.  202  [Jul>i, 

of  the  laboratory  is  not  harmful.  Even  exposure  to  direct  sunlight 
for  several  months  without  transfer  did  not  kill  organisms  when  grown 
upon  favorable  media  with  precautions  to  prevent  evaporation.  Under 
such  conditions  a  temperature  of  47 °C.  in  the  flask  was  reached  with 
the  thermometer  shaded. 

Slight  alkalinity  to  +20°  to  +25°  acid  (Fuller's  scale)  with 
phenolphthalein  is  tolerated;  neutral  to  -(-10°  is  best.  Growth  is 
generally  better  in  gelatin  or  agar  media  than  in  liquid  media  of  the 
same  composition. 

In  an  agar  stab  a  typical  drop-form  colony  is  produced  at  the  sur- 
face. A  thin,  gray  growth  follows  the  line  of  stab. 

MaltoSe  as  a  source  of  carbon  has  little  if  any  advantage  over 
saccharose  or  dextrose.  Mannite  is  also  suitable  as  a  source  of  carbon. 

In  standard  beef  broth  the  growth  of  the  organism  is  slow.  The 
liquid  becomes  cloudy,  a  gray-white  ring  is  formed,  and  a  thin  mem- 
brane covers  the  surface.  Later  a  flocculent  precipitate  settles  to  the 
bottom  of  the  tube. 

In  standard  beef-broth  gelatin  the  growth  of  the  organism  is  at 
first  funnel-shaped  and  then  stratiform.  The  gelatin  slowly  liquefies, 
the  process  sometimes  requiring  two  or  three  months  for  completion. 
In  gelatin  stabs  the  growth  sometimes  seals  over  the  stab  with 
a  drop-form  growth  and  liquefaction  does  not  occur.  If  inoculated 
tubes  are  kept  for  several  weeks  at  a  temperature  just  allowing  the 
gelatin  to  remain  liquid,  upon  cooling  it  will  be  found  that  the  gelatin 
refuses  to  solidify,  whereas  the  gelatin  in  uninoculated  check  tubes 
does  solidify.  The  enzyme  causing  liquefaction  is  present. 

On  ash-agar  plates  the  presence  of  Penicillium  glaucum,  which 
occasionally  intruded,  seemed  to  benefit  the  colonies  of  Ps.  radicicola 
that  were  in  close  proximity.  Ash  agar  upon  which  Penicillium 
glaucum  had  been  allowed  to  groAv  for  two  weeks  and  which  had  then 
been  sterilized  and  filtered,  had  a  noticeable  advantage  over  untreated 
ash  agar,  especially  with  the  slower  growing  organisms,  such  as  those 
of  Vigna,  Glycine,  and  Genista. 


Ti0J, 


PLATE  II 

Fig.  1. — Ash-agar  plate  from  pea  (Pisum  sativum},  seven  days  old 
Fig.  2. — Ash-agar  plate  from  dyer's  greenweed  (Genista  tinctoria),  twenty- 
five  days  old 


V 


x 


"2 
'3 


X 


•43 


a«=>. 

»  co 


/  * 


r  ^ 


.§  § 

:    O    O 

i   •<?»   r-a 

>? 


M 


> 
o  >» 


r_    ^.  .- 

ass 


mmm 


rH   N  CO 

bb  bb  bio 


N    ' 


f  » 


*     % 


t 


,      * 


•      > 


J 

-*         ~x* 


I  ,,.* 

,-• 


/    *      - 

-*     - 


.t 


-w 


^ 

»  r> 


i§ 

S?  10 


•*  o 

^•S 


93 

I  s 

'•  rO 

we 


>bo  s 

M          CO 


ll 


'       „        < 

^     / 


1: 

**      . 


111 

v  r^    ^ 


1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        125 

Part  II.— CROSS-INOCULATION:    VARIETIES  OF  NODULE 

BACTERIA 

CROSS-INOCULATION  INVESTIGATIONS 

It  was  early  recognized  that  certain  legumes  require  one  specific 
organism  for  inoculation.  For  example,  to  inoculate  soybeans  it  had 
been  found  necessary  to  import  soil  upon  which  soybeans  had  grown, 
as  the  bacteria  from  other  legumes  were  not  capable  of  causing  infec- 
tion. A  few  cross-inoculations  which  occur  under  field  conditions 
were  also  early  recognized.  The  bacteria  of  alfalfa  and  sweet  clover* 
were  known  to  be  identical,  as  were  those  of  the  cowpea  and  partridge 
pea.  A  third  group  the  bacteria  of  which  were  known  to  be  inter- 
changeable included  pea,  vetch,  sweet  pea,  and  lentil. 

Several  investigators,  notably  Laurent,14  Maze,21  Moore,28  and 
Kellerman,36  claimed  to  have  produced  cross-inoculations  which  do  not 
occur  naturally.  Little  credence  can  be  given  these  claims,  however, 
since  these  men  apparently  did  not  fully  appreciate  what  has  been 
frequently  referred  to  as  the  ubiquity  of  Ps.  radicicola.  No  doubt  their 
technic  was  at  fault. 

Methods  Used  in  Cross-Inoculation  Work. — For  testing  cross-in- 
oculations bacteria  were  isolated  from  as  many  genera  and  species  of 
legumes,  both  wild  and  cultivated,  as  could  be  obtained.  Great  care 
was  taken  in  their  isolation  and  in  the  maintenance  of  purity.  Two 
methods  of  testing  crosses  were  used — the  pot-culture  method  and  the 
agar  test-tube  method  of  Garman.38  In  the  pot-culture  method,  plants 
were  grown  in  one-gallon  pots  of  limed  white  quartz  sand  watered 
with  a  nutrient  solution  less  nitrogen,  as  described  by  Hopkins  and 
Pettit.b  The  sand  was  not  sterilized,  as  it  was  dry  and  clean,  and 
sterile  so  far  as  legume  bacteria  were  concerned,  as  proven  by  the 
record  of  the  check  pots.  The  pots  were  washed  clean  and  exposed 
to  sunlight  in  the  greenhouse  a  week  before  using,  being  turned  several 
times.  A  number  of  dry,  clean  pots  were  always  on  hand  so  that  no 


"Hopkins.2* 

Trom  Hopkins  and  Pettit  Laboratory  Manual  for  Soil  Fertility,  page  34. 

Solution  No.  1. — Nitrogen:  Dissolve  80  grams  of  ammonium  nitrate  in 
2500  cc.  of  distilled  water. 

Solution  No.  2. — Phosphorus:  Dissolve  25  grams  of  mono-calcium  phos- 
phate in  2500  cc.  of  ammonia-free  water. 

Solution  No.  3. — Potassium:  Dissolve  50  grams  of  potassium  sulfate  in 
2500  cc.  of  ammonia-free  water. 

Solution  No.  4. — Magnesium:  Dissolve  20  grams  of  magnesium  sulfate 
in  2500  cc.  of  ammonia-free  water. 

Solution  No.  5. — Iron:  Dissolve  .1  gram  ferric  chlorid  in  250  cc.  of 
ammonia-free  water. 

Use  10  cc.  each  of  Solutions  Nos.  1,  2,  3,  and  4,  and  1  cc.  of  Solution 
No.  5  per  liter  of  water.    When,  nitrogen  is  omitted  the  fact  is  so  stated. 


126 


BULLETIN  No.  202 


[July, 


1U17]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        127 

loss  of  time  resulted  when  organisms  were  to  be  tested.  The  seeds  were 
sterilized  by  shaking  them  violently  in  Harrison  and  Barlow's  steril- 
izing fluid,  previously  described,  allowing  them  to  remain  in  the  fluid 
for  ten  minutes,  and  then  washing  in  distilled  water.  Usually  five 
seeds  were  planted  in  a  pot.  Inoculation  was  made  at  the  time  of 
planting  by  adding  the  contents  of  an  agar  slant  mixed  with  sterile 
distilled  water.  One  pot  in  each  four  was  left  uninoculated  as  a  check. 

Occasionally  scattered  nodules  did  appear  on  checks  and  in  pots 
which  when  repeated  gave  negative  results.  The  use  of  open  pots  in 
a  greenhouse  frequented  by  many  people,  together  with  the  presence 
of  occasional  insects,  etc.,  cannot  but  result  in  some  chance  inoculations. 
A  chance  inoculation,  however,  is  easily  distinguished  from  a  true  one, 
for  in  the  former  case  the  nodules  are  few  in  number  and  widely  scat- 
tered, whereas  in  a  true  inoculation  the  nodules  are  numerous  and 
clustered  in  a  mass  about  the  tap  root.  This  pot-culture  method  was 
used  for  growing  plants  with  large  seeds,  such  as  Vigna,  Glycine, 
P'isum,  Vicia,  Lathyrus,  and  Phaseolus. 

In  the  second  method  used,  that  of  Garman,38  seeds  were  planted 
in  test-tubes  (6"  x  %")  containing  a  medium  composed  of  .65  percent 
agar  in  distilled  water.  No  nutrients  were  added.  The  agar  was 
inoculated  at  42°  to  45°C.  Seeds  (usually  three  to  a  tube),  sterilized 
as  before,  were  dropped  upon  the  agar  and  set  apart  with  a  flamed 
platinum  needle.  Generally  the  nodules  resulting  were  not  numerous, 
but  where  the  seeds  germinated  well,  results  were  always  positive  and 
dependable.  This  method  of  testing  crosses  is  especially  adapted  to 
smaller  seeds,  such  as  Melilotus,  Medicago,  and  Trifolium.  Large 
seeds  give  trouble,  as  they  are  difficult  to  sterilize. 

Vigna  X  Cassia. — The  inoculation  of  the  cowpea  by  bacteria  from 
the  partridge  pea  was  first  reported  by  Hopkins.34  In  the  other  cross- 
inoculations  mentioned  above  (alfalfa  and  sweet  clover;  pea,  sweet 
pea,  vetch,  and  lentil),  the  plants  having  a  common  organism  stand 
in  close  botanical  relationship,"  while  Vigna  sinensis  and  Cassia 
cliamaecrista  are  widely  separated.  Moreover,  the  former  is  a  plant 
introduced  from  Asia,  while  the  latter  is  a  native. 

The  first  cross-inoculation  experiments  in  these  investigations 
were  conducted  with  the  partridge  pea  (Cassia  chamaecrista) ,  inocu- 
lating it  as  shown  in  Table  3.  Partridge-pea  seeds  and  nodules  were 
obtained  from  plants  found  upon  virgin  prairie  in  a  wild  locality 
where  in  all  probability  cowpeas  had  never  been  grown.  Cultures  were 
obtained  from  cowpea  nodules  grown  in  the  greenhouse.  Thus  the 
sources  of  the  organisms  were  wide  apart. 

The  seeds  were  planted  on  October  16,  1915,  three  in  each  pot ;  the 
plants  were  examined  and  photographed  on  November  19  (see  Plate 
VI) .  The  results  appear  in  Table  3.  The  number  of  nodules  reported 


•Engler  und  Prantl:     Die  Naturlichen  Pflanzenfamilien,  III. 


128 


BULLETIN  No.  202 


\Jubj, 


1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        129 

may  be  somewhat  low,  as  it  is  difficult  to  count  the  smaller  nodules. 
The  checks  were  examined  with  great  care  and  found  to  be  free  from 
nodules. 


TABLE  3.  —  PARTRIDGE  PEA  x  COWPEA   (Cassia  chamaccrista  X 


sinensis} 


Pot 
No. 

Plant 

No.  of 
plants 

Source  of  inoculation 

Nodules 

Besults 
+  or- 

4593 
4594 
4595 
4596 

Partridge  pea 

II                    II 

tt           it 
it           ii 

3 
3 
3 
3 

Partridge  pea  No.  4608 
"          "No.  4609 
"          "No.  4611 
»          "No.  4613 

17,15,  9 
3,11,  7 
5,  5,  5 
3,  3,11 

4597 
4598 
4599 
4600 

Partridge  pea 

a             tt 

it             it 

3 
3 
3 
3 

Check 
it 

it 

it 

0,  0,  0 
0,  0,  0 
0,  0,  0 
0,  0,  0 

— 

4601 
4602 
4603 
4604' 

Partridge  pea 
it           » 

it           it 
it           it 

3 
3 
3 
3 

Cowpea  No.  4615 
"       No.  4617 
"       No.  4619 
"      No.  4621 

12,  3,  8 
8,10,  7 

7,  8,  6 
0,  0,  0 

"For  some  unknown  reason  the  plants  in  this  pot  produced  no  nodules. 

The  reciprocal  was  then  tried.  Five  seeds  of  cowpea  were  planted 
in  each  pot  and  inoculations  made  as  shown  in  Table  4.  The  seeds 
were  planted  on  November  29,  1915 ;  the  plants  were  examined  and 
photographed  on  January  3,  1916  (see  Plate  VII).  The  results  are 
also  shown  in  Table  4. 


TABLE  4. — COWPEA  x  PARTRIDGE  PEA  (Vigna  sinensis  x  Cassia  chamaecrista) 


Pot 

No. 

Plant 

No.  of 

plants 

Source  of  inoculation" 

Nodules 

Results 
-f-  or  — 

5023 
5024 
5025 
5026 

Cowpea 

ii 
a 

5 
5 
5 
5 

Cowpea  Nos.  4398  and  5042 
"      Nos.  4614    "    5039 
'       Nos.   4616     :  '    5040 
'      Nos.  4618     "    5041 

Abundant 
ii 

+ 
+ 

5027 
5028 
5029 
5030 

Cowpea 
it 

5 
5 
5 
5 

Cheek 

None 

— 

5031 
5032 
5033 
5034 

Cowpea 
a 

5 
5 
5 
5 

Partridge  pea  Nos.  4605  and  5035 
"          "    Nos.  4607     "    5036 
"    Nos.  4610     "    5037 
"          "    Nos.  4612     "    5038 

Abundant 

4- 
+ 

Vigna  X  Acacia. — Great  interest  had  been  taken  in  some  prelimi- 
nary trials  which  had  given  evidence  that  a  cross  exists  between 
cowpea  and  acacia.  Accordingly  cowpea  plants  were  inoculated  with 
cultures  from  six  species  of  Acacia,  and  later  with  a  culture  from  u 
seventh.  These  were  Acacia  armata,  floribunda,  linifolia,  longifolia, 
semperflora,  and  a  species  the  nodules  of  which  had  been  received  from 


130 


BULLETIN  No.  202 


[July, 


fcC 


1917]    POSSIBLE  SYMBIOSIS  BETWEKN  LEGUME  BACTERIA  AND  NON-LEGUMES        131 


California  but  of  which  nothing  else  was  known  except  that  it  is  an 
ornamental  tree.  Cultures  of  the  first  five  species  were  obtained  from 
nodules  of  plants  grown  in  the  horticultural  greenhouse  on  this  cam- 
pus. Cultures  from  a  seventh  species,  Acacia  melanoxylon,  which  was 
later  grown  in  this  greenhouse,  behaved  exactly  like  the  other  six. 
Seeds  were  planted  on  January  13,  1916;  the  plants  were  examined 
and  photographed  on  February  21  (see  Plate  VIII).  The  results  are 
shown  in  Table  5. 

TABLE  5. — COWPEA  x  ACACIA  (Vigna  sinensis  X  Acacia} 


Pot 
No. 

Plant 

No.  of 
plants 

Source  of  inoculation 

Nodules 

Results 
-for  — 

5464 
5465 
5466 

Cowpea 

5 
5 
5 

Acacia  armata  Nos.  5578  and  5649 
Acacia  floribunda  Nos.  5579  and  5650 
Acacia  linifolia  Nos.  5580  and  5651 

Abundant 

-- 

5467 
5468 
5469 
5470 

Cowpea 
tt 

it 

5 
5 
5 
5 

Check 

None 

— 

5471 
5472 
5473 

Cowpea 

5 
5 
5 

Acacia  longifolia  Nos.  5581  and  5652 
Acacia  semperflora  Nos.  5582  and  5653 
Acacia  ?  (from  California)  Nos.  5583 
and  5654 

Abundant 

•    + 
+ 

Cowpea  X  Several  Generic  Groups. — Tests  made  from  time  to 
time  with  the  cowpea  had  shown  that  infection  could  be  produced  with 
bacteria  from  eight  different  generic  groups  besides  the  cowpea  organ- 
ism. An  experiment  was  then  conducted  to  bring  together  the  results 
of  these  previous  trials.  The  results  are  given  in  Table  6. 

TABLE  6. — COWPEA  ( Vigna  sinensis)  x  SEVERAL  GENERIC  GROUPS 


Pot 
No. 

Plant 

No.  of 
plants 

Source  of  inoculation 

Nodules 

Results 
-f-or- 

6309 

Cowpea 

5 

Check 

None 

— 

6310 

5 

Acacia  (Acacia  melanoxylon) 

Abundant 

+ 

6311 

5 

Lead  plant  (Amorpha  canescens) 

None 

6312 

5 

Hog  peanut  (Amphicarpa  monoica) 

Several 

— 

6313 

5 

Check 

Several 

— 

6314 

5 

Peanut  (Arachis  hypogoea) 

Abundant 

-- 

6315 

5 

Wild  indigo   (Baptisia  tinctoria) 

Abundant 

-- 

6316 

5 

Partridge  pea   (Cassia  chamaecrista) 

Abundant 

— 

6317 

5 

Check 

None 

—  • 

6318 

5 

Tick  trefoil  (Desmodium  canescens) 

Abundant 

+ 

6319 

5 

Dyer's  greenweed  (Genista  tinctoria) 

Abundant 

6320 

5 

Japan  clover  (Lespedeza  striata) 

Abundant 

+ 

6321 

5 

Check 

None 

6322 

5 

Common  locust  (Eohinia  pseudo- 

acacia) 

Several 

— 

6323 

5 

Velvet  bean   (Mucuna  utilis) 

Abundant 

+ 

6324 

5 

Cowpea  (Vigna  sinensis) 

Abundant 

132 


BULLETIN  No.  202 


[July, 


PLATE  IX 

Seedlings  of  cowpea  (Vigna  sinensis)  inoculated  as  follows:  A. — Partridge 
pea  (Cassia  cliamaecrista) ;  B. — Tick  trefoil  (Desmodium  canescens)',  C. — Dyer's 
greenweed  (Genista  tinctoria) ;  D. — Check;  E. — Japan  clover  (Lespedeza  striata) ; 
F. — Velvet  bean  (Mucuna  utilis) ;  G. — Cowpea  (Vigna  sinensis);  H. — Check; 
I. — Acacia  (Acacia  melanoxylon) ;  J. — Peanut  (Arachis  hypogoea) ;  K. — Wild 
indigo  (Baptisia  tinctoria)  ;  L. — Check 


POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES 


In  Plate  IX  two  plants  from  each  pot  are  shown;  those  which 
were  negative  have  been  omitted.  The  results  confirmed  those  of  the 
earlier  tests.  The  plants  in  three  of  the  negative  pots  (those  crossed 
with  Amphicarpa,  Robinia,  and  a  check)  had  several  nodules,  but 
these  were  no  doubt  accidental  as  they  were  very  scattered.  In  a  pre- 
vious trial,  inoculations  with  Amphicarpa  and  Robinia  had  both  given 
negative  results. 

Lens  X  Several  Generic  Groups, — Another  set  of  similar  experi- 
ments is  of  interest.  Lentils  were  planted  on  March  17,  1916,  and 
inoculated  with  bacteria  from  several  generic  groups.  The  seedlings 
were  examined  on  April  14.  The  results  are  shown  in  Table  7. 

TABLE  7. — LENTIL  (Lens  esculenta)  x  SEVERAL  GENERIC  GROUPS 


Pot 

No. 

Plant 

No.  of 
plants 

Source  of  inoculation 

Nodules 

Results 
-for- 

6386 

Lentil 

3 

Scarlet  runner  bean   (Phaseolus  multi- 

florus) 

None 

— 

6387 

> 

3 

Common  bean  (Phaseolus  vulgaris) 

None 

— 

6388 

> 

3 

Trailing  wild  bean   (Strophostyles  hel- 

vola) 

None 

— 

6389 

i 

3 

Perennial  pea  (Lathyrus  latifolius) 

Abundant 

+ 

6390 

i 

3 

Common  garden  pea   (Pisum  sativum) 

Abundant 

6391 

»  . 

3 

Field  pea  (Piswm  arvense) 

Abundant 

-)- 

6392 

i 

3 

Broad  bean  (Vicia  faba) 

Abundant 

+ 

6393 

» 

3 

Check 

None 

In  a  similar  experiment  with  seedlings  of  Trigonella  foenum- 
graecum,  it  was  found  that  they  could  be  infected  with  the  bacteria 
from  Melilotus  alba  and  Medicago  sativa.  In  fact,  many  inoculation 
trials  were  made  with  all  available  bacteria.  Many  results  were  nega- 
tive, as  is  shown  in  Table  9. 

Tests  with  Garman's  Method. — The  cross-inoculation  trials  made 
by  Garman's  method  together  with  the  results  obtained  are  shown  in 
Table  8.  Four  inoculated  tubes  and  one  check  were  used  for  each  cul- 
ture tested.  In  Plate  X  are  shown  seven  cultures  tested  upon  Medicago 
sativa  (only  one  tube  of  each  of  the  seven  series  is  shown)  together 
with  two  checks.  In  all,  this  photograph  represents  thirty-five  tubes, 
twenty-eight  inoculated  and  seven  uninoculated. 

Results  of  Cross-Inoculation  Trials. — Table  9  gives  in  full  the 
results  of  the  cross-inoculation  experiments  conducted.  All  available 
cultures  were  tried  upon  seedlings  of  Anthyllis  vulneraria  and  Mimosa 
pudica,  but  no  nodules  were  produced.  It  is  assumed  that  the  organ- 
isms of  these  plants  are  distinct  from  any  of  those  used. 


134 


BULLETIN  No.  202 


11? 

O  *S    £ 

11                      III                      II 

*<3  ^  ^ 

1             1                      III                      II 

. 

lit 

"*  o''^ 

^  »<    53 

1 

i  KB 

f  JaSis 

1  1  I      I                1      !  !  +++++ 

i  + 

OQ  w  i3?  ^ 

1 

5 

« 
o 

5 

o 
c4  21 

i 

"3  1 

]      1                      1    ,1  I  +++++ 

i  + 

w 

p—  H    §   «*S 

s 

CO 

«*£§ 

"fc 

O     x-N 

a 

^     F*(J      ^i     Q 

s 

3 

o 

"f*1 

1                      III  +++++ 

i  + 

tM 

v.x'^' 

« 

c3   ^  ^^^ 

co 

®  tS   y 

ATION 

1  1/5 

si4? 

+    1  ++++++    1  ++]              M 

1  1  + 

g 

H-S-_ 

S 

IH 

i 

W 

<» 

CO 

• 

oa 

Q                            « 

o 

53                       2    ^ 

Hi 

H 

V                         Q      ^      eS                                   G 

O       ^**                                  ^i 

f 

od 

R 

•a 

.2 

liiil'lill^llllii^ll. 

.2       E  §  «  S 
*~       «  'S  s  § 

«     _>     A    rfi     ^     1 

•I  1  i  »  1  1  -1 

H 

P3      *^    53    ^3    S^j  si    S                ^5   ^j7^    5^    GO    ^-    5S    ^  r^   ?£  ^^1*" 

^    ?^    S    ^    Sj**^*  to    5^ 

9 

03 

"*?              O                 i^    .C*     "*"*      S      ••      ••    *«*       r*                 S       S      S5j     ^.       ^       .                               S 

Cn    ti      S    ^^    c*      ^3    ri^      SS 

•4 

H 

a 

o 

•M 

*M 

^ 
3 
E 

O 

w 

o 

^^^^PQ^I^QCb^JK^I^    K^K^^^^^^S^ 

BH^fS^QHtH^-^ 

.2 

0 

1 

® 

01 

o 

E 

S           S3 

§ 

® 

•8        o 

CO 

02 

1 

cd            r^J 

o 

H 

3            «   ^>    4) 

r2     0 

g 

T3                                          .              >    01    f* 
§0            O    OJ    O 

o  £ 

o 

0      13  ^  o 
Co              P-                               i^              co 

g 

g 

a>            p                 *<        g       .p       -g 

^  .  i 

o 
O 

^P      'o'og        ^^feQo>        ^oSrt              «fef-'rMii 

Jis3|1lllMpts!t||    !.|||:s 

rt^  M^St^'^'^  OS'S.*  ^  0,2  s  ^  '"^  2  s  ^ 

ctf    c^   ^    rt  "Ti    fl  "r3    P 

•^^Wpn^pHE-iEHQc/JCC^i-s^^-!-^^^^^ 

W^SHMf^Wo 

1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        135 


(sapads 


I  I 


I  I 


I  I  I I++I    I  I  I  I  1  + 


(sapads  XTS)  wmjo/uj 


I  I  I  I  I  I  I  I  I  I  I  I    I  I  I l+l 


+ 


moiooD-opndsd  wimqog 


I  I  I  II  I  I  I  I 


I 


wnaiivs  PUB  9suMj,v  wrasjj 


I  l+l 


(sapads 


vunonjy 


I  I  I  l+l  I  M  I  I  I 


(sapads  aaiqi) 


I  I  I  .1+1  I 


(sapads  Jnoj) 


I  I 


I  I  I 


snuidn/2 


I  I  I  I  I  I  l+l  I  I     +1 


PUB 


II     I  I  I  I  I  I 


I  I  I  I  I  I ++ 1  I  I  I  I  I  I  I + 1 


sniio.fij.vi  PUB 


SU90S9UVO 


oissvg 


SU90S9UV9 


I  I 


(sapads  uaAas)  movoy 


uopB{ii3oni  jo  aoinog 


Botani 
name 


e 


g 


-     -  g  rf- 


it! 

o-5  s  rg  § 

t       J 


l 

-  • 


tniif     -t 

i  sat  ii  ni  ill  a  <il  §1 


136  BULLETIN  No.  202 

Based  upon  the  trials  made,  the  nodule  organisms  are  divided 
into  the  following  groups  according  as  they  are  interchangeable  for  the 
purposes  of  inoculation : 

GROUP  I 

Mammoth  red  clover,  Trifolium  pratense  perenne 

Alsike,  or  Swedish  clover,  Trifolium  hybridum 

Crimson  clover,  Trifolium  incarnatum 

Berseem,  or  Egytian  clover,  Trifolvum  alexandrianum 

White  clover,  Trifolium  repens 

Zigzag,  or  cow  clover,  Trifolium  medium 

GROUP  II 

White  sweet  clover,  Melilotus  alba 

Yellow  sweet  clover,  Melilotus  officinalis 

Wild  yellow  sweet  clover,  Melilotus  indica 

Alfalfa,  Medicago  sativa 

Alfalfa,  Medicago  falcata 

Bur  clover,  Medicago  hispida 

Black  medick,  or  yellow  trefoil,  Medicago  lupulina 

Fenugreek,  Trigonella  foenum-grae<yum 

GROUP  III 

Cowpea,  Vigna  sinensis 

Partridge  pea,  Cassia  chamaecrista* 

Peanut,  Arachis  hypogoea 

Japan  clover,  Lespedesa  striata 

Slender  bush  clover,  Lespedesa  virginica 

Velvet  bean,  Mucuna  utilis 

Wild  indigo,  Baptisia  tinctoria 

Tick  trefoil,  Desmodium  canescens 

Tick  trefoil,  Desmodium  illinoense 

Acacia,  Acacia  armata 

Acacia,  Acacia  floribunda 

Acacia,  Acacia  lini folia 

Acacia,  Acacia  longifolia 

Acacia,  Acacia  melanoxylon 

Acacia,  Acacia  semperflora 

Acacia,  Acacia  ?,  from  California 

Dyer's  greenweed,  Genista  tinctoria 

GROUP  IV 

Common  garden  pea,  Pisum  sativum 

Field  pea,  or  Canada  field  pea,  Pisum  sativum  arvense 

Hairy  vetch,  Vicia  villosa 

Spring  vetch,  Vicia  sativa 

Broad  bean,  Vicia  faba 

Narrow  leaved  vetch,  Vicia  angustifolia 

Vetch,  Vicia  daysiecarpa 

Lentil,  Lens  esculenta 

Sweet  pea,  Lathyrus  odoratus 

Perennial  pea,  Lathyrus  latifolius 


•Cultures  isolated  from  nodules  of  Cassia  nictitans  and  tested  on  seedlings  of 
Cassia  chamaecrista  and  Vigna  sinensis  failed  to  produce  nodules.  The  cultures 
had  been  on  hand  some  time  when  tried,  and  it  was  suspected  that  an  error  had 
been  made.  At  a  later  time  a  number  of  seedlings  of  Cassia  medsgeri  were  grown 
and  inoculated  with  the  Cassia  nictitans  cultures,  as  well  as  with  bacteria  from 


1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        137 

GROUP  V 
Soybean,  Glycine  hispida 

GROUP  VI 

Garden  bean,  Phaseolus  vulgaris 
Garden  bean,  Phaseolus  angustifolia 
Scarlet  runner  bean,  Phaseolus  multiflorus 

GROUP  VII 

Lupine,  Lupinvs  perennis 
Serradella,  Ornithopus  sativus 

GROUP  VIII 
Hog  peanut,  Amphicarpa  monoica 

GROUP  IX 
Lead  plant,  Amorpha  canescens 

GROUP  X 
Trailing  wild  bean,  Strophostyles  helvola 

GROUP  XI 
Black,  or  common  locust,  Eobinia  pseudo-acacia 

GROUPING  BY  SEROLOGICAL  TESTS  AND  BY  CULTURAL  DIFFERENCES 

Review  of  Results  with  Serological  Tests. — In  order  to  throw  light 
upon  the  kinship  among  the  various  nodule  bacteria,  Zipfel,37  in  1912, 
made  use  of  the  agglutination  method.  From  his  results  he  concluded 
that  the  nodule  bacteria  were  not  varieties  of  the  same  species,  but 
that  distinct  species  existed. 

Klimmer  and  Kriiger39  two  years  later  used  serological  tests  to 
distinguish  species.  They  used  the  agglutination  method  principally ; 
and  complement-binding  and  precipitation  for  confirmation  and  con- 
trol. Working  with  organisms  from  eighteen  legume  species,  they 
divided  the  bacteria,  according  to  their  methods,  into  nine  species, 
which  they  asserted  differed  sharply  from  one  another. 

Simon,40  in  1914,  tested  various  cultures  upon  seedlings  of  several 
legume  species,  and  compared  the  results  with  those  obtained  by  using 
Zipfel's  agglutination  method.  He  found  that  the  results  of  both 
methods  agreed  substantially.  His  grouping  of  the  nodule  bacteria 
is  in  general  agreement  with  that  of  Klimmer  and  Kriiger."  He  con- 
cluded, however,  that  "the  root  bacteria  of  legumes  are. rather  to  be 
conceived  as  more  or  less  constant  adaptations  of  the  species  Bacillus 
radicicola." 


Cassia  chamaecrista  and  Vigna  sinensis,  but  no  nodules  were  produced.  This 
suggested  that  perhaps  there  is  more  than  one  adaptation  affecting  Cassia.  The 
evidence  is  not  conclusive,  however. 

•It  should  be  noted  that  the  pure  cultures  used  by  Klimmer  and  Kriiger  were 
supplied  by  Simon. 


133 


BULLETIN  No.  202 


[July, 


_to 

a 

ce 

E 

>-i 

a 

O 

h 

O 

<H 

CS 

in 

-t^    3 

t*  ^ 

*   § 

03  "3 

C    'i 

•S  a> 

5 

fl  « 
^  * 

O    r*t 

M 

^g 

!     g 

^•js 

•§4S 

•4 
J 

PM 

e  S 

"    0 

o.S 

o  t>c 

•2  2 

II 

ctf  -4-j 

<H    « 

13  S 

•H 

O 

O3 

fcJD 

•  pH 

'S 

v 

V 

1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        139 

Grouping  ~by  Cultural  Differences. — While  no  serological  tests 
were  made  in  these  experiments,  a  possible  basis  of  distinguishing 
varieties  or  species  was  observed  in  the  striking  differences  among  the 
organisms  upon  culture  media.  When  grown  upon  ash-agar  slants, 
three  quite  distinct  types  of  growth  were  noted.  The  organisms  were 
divided  upon  this  basis  into  three  groups  as  follows : 

Group  I. — The  organisms  are  distinguished  by  the  thin,  scant 
growth  upon  the  slant ;  the  streak  is  a  dull  gray- white.  They  are 
exceedingly  slow  growers,  colonies  on  agar  plates  being  especially  slow. 
They  are  not  sticky  to  the  touch,  and  spread  quite  easily  in  water  upon 
the  cover  slip.  Flagella  are  quite  easily  demonstrated,  since  there  is 
little  gum  to  interfere.  The  group  includes  Vigna,  Cassia,  Acacia, 
Lcspedeza,  Desmodium,  Baptisia,  Genista,  Arachis,  Mucuna,  Glycine, 
and  Amphicarpa. 

Group  II. — The  organisms  of  this  group  grow  more  rapidly  than 
those  of  Group  I.  The  growth  is  moderate  to  abundant  with  but  little 
tendency  to  spread.  The  streak  is  raised,  glistening,  opaque,  and 
pearly  white  in  color.  Tho  not  usually  very  sticky  to  the  touch,  there 
is  considerable  gum,  which  seriously  interferes  in  attempts  to  stain 
flagella.  Melilotus,  Medicago,  and  Trigonella  make  up  this  group. 

Group  III. — The  growth  of  these  organisms  is  very  fast,  and  there 
is  a  strong  tendency  to  spread.  The  streak  is  watery  and  semi-translu- 
cent, being  quite  different  from  the  opaque  growth  of  Group  II.  The 
surface  is  not  so  shiny  as  in  that  group.  Further,  the  organisms  are 
quite  slimy  and  usually  quite  sticky  to  the  touch.  The  excessive  amount 
of  gum  prevents  staining  of  flagella  and  holds  the  organisms  in  clumps 
so  that  they  cannot  be  spread  easily  on  the  cover  slip.  The  organisms 
of  Vicia,  Pisum,  Lens,  Latliyrus,  Trifolium,  Phaseolus,  and  Stroplio- 
styles  are  included  in  this  group. 

While  the  descriptions  above  apply  more  directly  to  growth  upon 
ash  agar,  the  differences  hold  true  for  growth  on  other  agar  media, 
especially  Fred's  synthetic  and  Conn's  asparaginate  agar.  The  latter 
is  especially  suited  to  the  organisms  of  Group  I,  growth  upon  it  being 
considerably  better  than  upon  ash  agar.  It  was  further  noted  that  these 
differences  did  not  disappear  with  the  aging  of  the  cultures,  but  on 
the  contrary  became  more  pronounced.  Cultures  which  were  two  years 
old  showed  in  young  transfers  the  differences  noted  in  freshly  isolated 
cultures. 

Varieties  vs.  Species. — While  the  grouping  of  the  various  nodule 
bacteria  is  perhaps  best  determined  by  actual  plant  inoculations,  yet 
the  results  of  serologic  tests  show  that  the  various  organisms  are 
different  and  that  these  differences  are  permanent.  Likewise  in  cer- 
tain cultural  characteristics  herein  described  we  find  differences  which 
also  are  permanent.  Furthermore,  the  adaptations  as  tested  by  actual 
inoculations  upon  plants  are  constant.  For  example,  the  soybean 


140  BULLETIN  No.  202  [July, 

organism  not  only  retains  its  individuality  as  tested  by  serologic 
methods  and  by  cultural  characteristics,  but  it  also  retains  its  special 
adaptation  to  the  soybean  plant,  in  spite  of  imposed  conditions 
designed  to  break  this  adaptation.  These  facts  form  perhaps  a  legiti- 
mate basis  for  the  belief  that  distinct  species  exist  among  the  nodule 
bacteria.  In  numerous  other  characteristics,  however,  these  bacteria 
are  so  much  alike,  and  as  a  whole  they  differ  so  widely  from  any  other 
species  of  bacteria,  that  it  seems  more  consistent  to  regard  the  adapted 
forms  as  varieties  of  the  single  species  Pseudomonas  radicicola. 

Experiments  in  cross-inoculation  brought  out  the  fact  that  in 
many  cases  in  which  a  single  organism  is  capable  of  infecting  several 
plant  genera,  the  host  plants  stand  in  close  botanical  relationship.  In 
Group  III,  however  (see  page  136),  is  a  striking  exception  to  this.  In 
Acacia,  Cassia,  and  Vigna,  we  have  each  of  the  three  sub-families  of 
the  Leguminosae  represented,  yet  the  same  organism  produces  nodules 
upon  all  three.  Obviously  botanical. relationship  is  not  responsible  in 
this  case. 

Maze21  claimed  that  the  reaction  of  the  soil  was  responsible  for 
the  special  adaptations.  He  divided  the  nodule  bacteria  into  two 
groups,  those  infecting  plants  which  have  become  accustomed  to  acid 
soil  and  those  infecting  plants  which  have  become  accustomed  to 
alkaline  soil.  By  gradually  accustoming  a  bacterium  from  the  alkaline- 
soil  group  to  an  acid  medium,  he  claimed  to  have  so  modified  it  that 
it  would  produce  nodules  upon  lupines,  which  belonged  to  his  acid- 
soil  group.  However,  observations  do  not  bear  out  Maze's  statements 
in  regard  to  the  two  groups.  The  reaction  of  the  soil  does  not  appear 
to  have  any  significance  in  determining  the  groups,  of  which  there  are 
certainly  more  than  two.  Furthermore,  the  reaction  of  artificial  media 
does  not  break  or  change  the  special  adaptations,  nor  is  the  organism 
modified  at  all  so  far  as  its  power  to  produce  nodules  is  concerned. 

Evidently,  then,  the  adaptation  is  between  the  root-sap  of  the 
plant  and  the  bacteria.  It  may  be  a  case  of  specific  enzymes  produced 
by  the  bacteria,  or  of  differences  in  the  root-sap  which  cannot  be 
detected  by  chemical  methods. 


1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  ANE  NON-LEQUMFS        141 

Part  III.— HISTOLOGY  OF  THE  NODULES  OF  THE 
LEGUMINOSAE 

Technic. — Sections  were  made,  for  the  most  part,  from  nodules  of 
plants  grown  in  normal  soil,  for  it  was  desired  to  study  especially  the 
nodules  as  they  occur  in  nature.  When  sections  from  young  nodules 
were  desired,  however,  plants  were  grown  in  quartz  sand,  watered  with 
the  nutrient  solution.* 

The  paraffin  method  of  imbedding  was  used  entirely.  Sections 
were  usually  cut  five  or  six  microns  thick ;  thinner  sections  were  tried 
but  abandoned  owing  to  the  difficulty  in  getting  good  mounts.  In  the 
earlier  attempts,  material  was  fixed  by  immersing  from  four  to  six 
hours  in  a  picric  fixing  agent."  Haidenhain's  iron-haematoxylin  was 
usually  used  in  conjunction  with  the  picric  fixative.  Sections  were 
mounted  in  series  and  stained  upon  the  slides,  after  removing  the  par- 
affin with  xylol,  etc.  The  mounts  obtained  were  not  entirely  satis- 
factory, but  were  valuable  for  comparison  with  mounts  stained  other- 
wise. Differentiation  with  the  ferrous-ammonium-sulfate  solution  can 
bo  carefully  controlled,  which  is  the  great  advantage  of  this  method. 

Flemming's  method  was  later  adopted  for  most  of  the  work. 
Nodules  were  fixed  in  Flemming's  weaker  solution  (chrom-osmic-acetic 
solution),  and  the  triple  stainc  then  applied.  Sometimes  the  triple 
stain  was  used  with  material  fixed  in  picric  acid,  and  altho  results  were 
not  so  good  as  with  material  fixed  in  the  chrom-osmic-acetic  fluid,  still 
very  satisfactory  mounts  were  obtained.  With  haematoxylin  mounts, 
cedar  oil  was  used  for  clearing ;  with  the  triple  stain,  clove  oil  was  pre- 
ferred. 

Considerable  difficulty  was  experienced  with  thin  sections  when 
the  triple  stain  was  used,  owing  to  the  rapid  loss  of  stain  when  dehy- 
drated with  ethyl  alcohol.  Dehydration  and  differentiation  with  ethyl 
alcohol  was  not  wholly  unsatisfactory ;  however,  by  using  amyl  alcohol 
instead  of  ethyl  alcohol,  more  densely  stained  mounts  were  obtained. 

For  demonstrating  starch,  slides  were  stained  with  Flemming's 
triple  stain  and  differentiated  with  ethyl  alcohol,  removing  all  excess 
stain.  They  were  then  transferred  to  distilled  water  to  remove  the 
alcohol,  after  which  they  were  mounted  in  distilled  water  to  which  a 
small  quantity  of  Lugol's  iodine  solution  was  added.  The  iodine  was 
not  concentrated  enough  to  discolor  the  field,  but  gave  the  character- 
istic blue  to  the  starch.  Water  was  added  during  examination  when 


"See  footnote,  page  125. 

"The  solution  was  made  up  as  follows : 

Corrosive   sublimate 5  gins. 

Glacial  acetic  acid 5  cc. 

Saturated  solution  of  picric  acid  in  70  percent 

alcohol 100  cc. 

°Safranin,  gentian-violet,  orange  G. 


142  BULLETIN  No.  202  [July, 

necessary  to  prevent  drying  out.  Such  mounts  are  not  permanent,  but 
they  may  be  made  so  by  restaining  and  mounting  in  balsam,  tho  the 
blue  color  is  not  retained  by  the  starch. 

A  useful  modification  of  Flemming's  triple  stain  is  to  follow  the 
gcntain-violet  with  Lichtgriin*  instead  of  orange  Gr. 

ORIGIN  OF  THE  NODULE 

Whether  the  active  motility  of  Ps.  radicicola  in  certain  stages  has 
any  bearing  ripon  the  infection  of  the  root-hairs,  or  young  roots,  of 
legumes,  is  not  known.  The  vigorous  root  system  of  the  Leguminosae 
in  general,  however,  is  probably  a  big  factor. 

The  entrance  of  the  organism  into  the  root  thru  the  root-hair,  as 
described  by  previous  investigators,  was  not  studied  in  these  investiga- 
tions. According  to  these  workers,"  the  infection  first  appears  as  a 
bright  spot  at  or  near  the  tip  of  the  root-hair.  The  bacteria  gain 
entrance  by  dissolving  the  cell  wall,  probably  thru  the  agency  of  an 
enzyme.  Infection  is  followed  by  a  distinct  bending  of  the  root-hair, 
the  response  to  the  irritation  set  up.  The  organisms  multiply  and 
form  a  zoogloeal  strand,  which  makes  its  way  down  the  root-hair  into 
the  root-cortex.  In  the  innermost  layers  of  the  cortex,  the  irritation 
is  set  up  which  gives  rise  to  the  nodule.  The  cells  are  stimulated  to 
rapid  growth,  a  meristem  is  formed,  and  the  young  nodule  emerges 
from  the  root  epidermis  as  a  mass  of  parenchymal  cells.  In  origin, 
then,  the  nodule  is  similar  to  the  lateral  root,  but  here  the  similarity 
ends. 

While  it  is  well  established  that  infection  takes  place  in  this  way, 
still  it  is  evident  that  it  is  not  thru  the  root-hairs  alone  that  the  bacteria 
gain  entrance.  The  root  epidermis  itself  may  be  penetrated,  as  with 
seedlings  grown  in  agar  test-tube  cultures  (Garman's  method),  in 
which  case  no  root-hairs  are  produced.  The  same  is  found  true  of 
seedlings  grown  in  sand  saturated  with  a  nutrient  solution  and  not 
inoculated  until  the  roots  have  made  several  inches  of  growth.  On 
allowing  time  for  infection  and  nodule  production,  it  will  be  found 
that  nodules  are  produced  abundantly  around  the  tap  root  above  the 
region  of  root-hairs. 

STRUCTURE  or  THE  NODULE 

In  Plate  XI  is  shown  the  gross  structure  of  nodules  of  Trifolium 
pratense  which  are  well  developed  but  still  growing.  Fig.  1  represents 
a  longitudinal  section  near  the  central  part  of  a  nodule.  The  greater 
portion,  it  will  be  seen,  is  made  up  of  bacteroidal  cells,  which  occupy 


•.5  gram  in  200  cc.  of  alcohol. 

"Marshall  Ward,"  "  and  Peirce23  should  be  consulted.  Other  important  contri- 
butions are  the  following.  Woronin,1  *  Eriksson,*  Frank,4  Beyerinck,9  Tschirch,1 
Vuillemin,11  Prazmowski,6  a  1!  Schneider,"  Atkinson,"  and  Dawson.24 » 


PLATE  XI 

Fig.  1. — Longitudinal  section  of  a  nodule  of  red  clover  (Tri folium  pratense) 
which  was  well  advanced  but  still  growing,  showing  vacuolated  bacteroidal  cells, 
vascular  tissue,  nodule  cortex,  and  meristem.  Stained  with  Flemming's  triple  stain 
with  amyl  alcohol  dehydration  X  100 

Fig.  2. — Cross-section  of  a  similar  nodule  of  red  clover  (Trifolium  pratense). 
Stained  with  Flemming's  triple  stain  and  mounted  in  dilute  iodine  solution  to  show 
the  starch.  The  fibro-vascular  bundles  are  especially  prominent  X  100 


'§  S 


M     O    f? 


•c  s 

2   03 


f  V£ 

02    02     O 


i-l  <M   CO 

bb  bio  bb 


*>  ^^  ~: 

:;...,  i  .     iHIUHlBfc 


PLATE  XIII 

Fig.  1. — Young  infected  cells  of  a  nodule  of  hairy  vetch  (Vicia  villosa) 
(same  as  Fig.  2,  Plate  XII).  Stained  with  Flemming's  triple  stain.  The  bacteria 
do  not  show  distinctly  X  430 

Fig.  2. — Bacteroidal  cells  of  red  clover  (Trifolium  pratense)  in  a  well  ad- 
vanced but  growing  nodule.  Cells  have  become  vacuolated  and  filled  with  bac- 
teroids.  Flemming's  triple  stain  with  amyl  dehydration  X  1080 


PLATE  XIV 
"Root  nodules  of  Ceanothus  americanus 


1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        143 

the  middle  area.  The  fibro- vascular  system  is  immediately  outside  this, 
inclosed  in  the  cortex  layer  of  cells.  At  the  tip  is  a  well  defined  meris- 
tcm  made  up  of  small,  rapidly  dividing  cells,  containing  large  nuclei. 
Few  bacteria  or  bacteroids  are  found  in  the  meristem  or  in  the  cortex ; 
they  are  confined  mostly  to  the  bacteroidal  cells.  Aside  from  possess- 
ing bacteroidal  tissue,  the  nodule  differs  from  the  lateral  root  in  that 
it  has  no  central  cylinder.  Further,  it  has  no  root-cap  and  no  epider- 
mis, but  is  inclosed  by  a  protective  band  of  corky  cells. 

Fig.  2,  Plate  XI,  shows  a  cross-section  of  a  nodule  of  Trifolium 
pratense  at  about  the  same  stage  of  development  as  that  shown  in  Fig. 
1.  This  section  was  stained  with  Flemming's  triple  stain  and  then 
mounted  in  dilute  iodine  solution  in  order  to  show  the  location  and 
distribution  of  starch.  The  fibro-vascular  bundles  may  be  seen  espe- 
cially well. 

The  bacteroidal  cells  of  younger  nodules  are  strikingly  different 
from  those  of  older  ones.  In  the  young  nodule  the  cells  are  full  and 
closely  packed.  Plate  XII  shows  sections  of  a  young  nodule  of  Vicia 
villosa.  Fig.  1  of  this  plate  shows  the  meristem,  and  Fig.  2  a  section 
some  distance  back.  The  latter  figure  should  be  compared  with  Fig. 
2  of  Plate  XI.  These  young  infected  cells  are  shown  more  highly 
magnified  (  X  430)  in  Plate  XIII,  Fig.  1.  The  cells  are  filled  with 
cytoplasm  in  which  are  dispersed  myriads  of  bacteria.  These  bacteria  • 
occur  chiefly  as  swarmers  or  as  small  vacuolated  rods,  so  that  it  is  diffi- 
cult to  resolve  them  in  the  cytoplasm.  The  nuclei  are  quite  prominent. 

As  the  nodule  becomes  older,  the  bacteria  are  more  in  evidence, 
the  bacteroids  becoming  especially  large  and  numerous.  The  nucleus 
becomes  distorted  and  is  pushed  to  one  side  of  the  cell,  tho  sometimes 
it  disintegrates  and  disappears  entirely,  giving  way  to  a  large  central 
vacuole,  which  is  inclosed  by  a  band  containing  mostly  bacteroids. 
Fig.  2,  Plate  XIII,  shows  a  few  cells  ( X  1080)  in  which  this  has  taken 
place.  (This  figure  shows  a  few  cells  of  Fig.  1,  Plate  XI,  more  highly 
magnified. )  It  is  in  this  stage  of  development  that  the  large,  branched 
bacteroids,  such  as  those  shown  in  Plate  III,  are  found. 

The  fibro-vascular  system  extends  from  the  meristem  region  to 
the  base  of  the  nodule,  where  the  elements  unite  and  communicate 
with  the  central  cylinder  of  the  lateral  root.  As  the  nodule  becomes 
older,  the  bacteria  further  devastate  the  cells  and  probably  automat- 
ically shut  off  the  food  supply  from  the  root,  whereupon  the  nodule 
decays  and  sloughs  off. 

The  so-called  infection  threads  (F'dden,  of  Tschirsch;  Infektion- 
schlauche,  of  Prazmowski)  so  frequently  found,  especially  in  young 
nodules,  were  the  objects  of  much  study.  Contrary  to  the  opinions  of 
Dawson23  and  Peirce,25  the  infection  threads  are  not  zoogloeal  strands 
made  up  of  small  bacilli,  but  are  solid  hyphae-like  structures  bearing 
a  remarkable  resemblance  at  times  to  tubes,  which  in  fact  some  earlier 


144  BULLETIN  No.  202  [July, 

investigators  believed  them  to  be  (see  Plate  XII,  Fig.  3).  No  septae 
were  found.  The  threads  were  more  frequently  seen  in  the  meristem 
or  in  the  cortex  cells  near  the  apex  of  the  nodule.  The  longest  one 
observed  in  a  single  section  traversed  six  consecutive  cells.  Shorter 
threads  were  frequently  encountered  in  the  bacteroidal  tissue.  Fre- 
quently the  threads  were  found  branched,  and  invariably  they  were 
growing  directly  toward  the  cell  nucleus  or  sending  a  branch  to  it.  In 
passing  thru  the  cell  walls  the  thread  becomes  peculiarly  thickened 
or  flattened,  producing  a  funnel-like  appearance.  This  also  occurs 
when  the  thread  approaches  the  nucleus. 

With  the  view  that  the  infection  threads  are  not  zoogloeal  strands 
composed  of  separate  bacilli,  they  become  more  difficult  to  explain.  A 
possibility  is  that  they  are  due  to  unusually  stimulated  bacteroids  or  to 
a  number  of  bacteroids  which  fail  to  divide  but  remain  attached  with 
the  resorption  of  the  cell  wall  between.  However,  this  is  pure  spec- 
ulation. 


1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        145 

Part  IV.— NON-LEGUMES  SAID  TO  BE  CONCERNED  IN  THE 
FIXATION  OF  ATMOSPHERIC  NITROGEN 

HISTORICAL 

The  demonstration  by  Hellriegel  and  Wilf arth5  10  of  the  fixation 
of  nitrogen  by  legume  plants  and  the  isolation  by  Beyerinck  of  the 
organism  from  the  legume  nodules,  stimulated  interest  in  the  root 
nodules  found  upon  non-legume  plants.  Of  the  groups  of  non-legumes 
which  possess  these  structures,  those  which  have  received  the  most 
attention  are  Ceanothus,  Elaeagnus,  Alnus,  Podocarpus,  Cycas,  and 
Myrica. 

The  earlier  investigators8  for  the  most  part  held  that  nodules  were 
of  fungous  origin.  Arzberger49  held  that  the  causal  agents  (Frankia 
ceanotlii  and  Frankia  subtilis)  in  CeanotJius  and  Elaeagnus  were  quite 
similar,  but  that  that  (Frankia  bruncJiorstii)  of  Mryica  was  quite  dif- 
ferent, being  of  the  nature  of  an  Actinomyces. 

Previous  to  Arzberger,  Hiltner45  in  1896  claimed  a  fixation  of 
nitrogen  by  Alnus  and  Elaeagnus.  In  1899,  Nobbe  and  Hiltner46 
claimed  the  same  for  Podocarpus,  the  nodules  of  which  were  said  to  be 
due  to  an  endotrophic  mycorrhiza. 

Bottomley48  b  claims  to  have  demonstrated  in  1907  the  presence  of 
nitrogen-fixing  bacteria  in  the  nodules  of  Cycas.  In  1912  the  same 
writer51  reported  the  isolation  of  an  organism  identical  with  Ps.  radici- 
cola  from  Myrica  gale  and  claimed  fixation  of  nitrogen  by  young 
Myrica  plants. 

In  the  same  year  Spratt,52  working  in  Bottomley 's  laboratory, 
reported  a  similar  isolation  from  Alnus  and  Elaeagnus  and  also  from 
Podocarpus.53 

More  recently  Bottomley54  has  reported  the  isolation  of  Ps.  radici- 
cola  from  CeanotJius,  and  shown  the  fixation  of  nitrogen  in  culture 
solutions  by  the  organism  isolated. 

CEANOTHUS  AMERICANUS 

Attempts  to  Isolate  tlie  Causal  Organism. — As  CeanotJius  ameri- 
canus  grows  close  at  hand,  material  for  study  was  easily  obtained. 
Efforts  to  isolate  a  causal  organism  were  persistent,  covering  a  period 
from  early  spring  to  late  fall.  Nodules  in  all  stages  were  plated,  spe- 
cial effort  being  made  upon  the  extremely  young  ones.  With  ash  agar 
alone  ninety  plates  were  poured  in  duplicate.  Legume  nodules  were 
frequently  plated  as  checks  upon  the  method,  the  same  procedure  being 
used  in  each  case.  Legume  nodules  nearly  always  gave  good  plates ; 
Ceanofhus  nodules  failed  always.  Variation  in  the  seeding  of  the 


•For  a  review  of  the  subject  Arzberger  should  be  consulted. 

"The  paper  written  in  1907  to  which  Bottomley  refers  was  not  found. 


146  BULLETIN  No.  202  [July, 

plates  was  tried.  Sometimes  the  entire  nodule  was  crushed  with  sev- 
eral cubic  centimeters  of  sterile  water  and  poured  with  the  plate ;  some- 
times several  loops  of  infusion  were  used ;  and  sometimes  the  nodules 
were  cut  open  and  the  tissue  scraped  out  for  plating.  Ash-agar  plates 
were  inoculated  direct  with  nodule  tissue  and  with  crushed  infusion. 

Other  media  were  tried.  The  list  included  Fred's  agar  (No.  201), 
Ashby's  (No.  202),  Spratt's  (No.  203),  beef -broth  agar  (No.  205), 
Conn's  asparaginate  agar  (No.  204),  Ceanothus-extract  agar  (simi- 
lar to  No.  206),  a  mixture  of  Ceanothus- extract  agar  and  ash 
agar,  potato  agar,  oatmeal  agar,  cornmeal  agar,  Loeffler's  blood- 
serum  agar,  and  Koch 's  blood-serum  agar.  Many  plates  were  poured 
and  many  direct  slants  tried.  Liquid  media  were  not  extensively  em- 
ployed as  this  means  of  isolation  is  objectionable.  However,  Spratt's 
medium  (No.  103)  and  beef  broth  (No.  105)  were  tried. 

In  no  case  did  a  typical  plate  resembling  those  obtained  in  plating 
legume  nodules  result.  For  the  most  part  the  plates  were  blank  except 
for  an  occasional  mold  or  yeast.  Bacterial  colonies  sometimes  grew, 
but  never  did  a  single  organism  persist  that  upon  examination  in  any 
way  resembled  Ps.  radicicola. 

Almost  invariably  slants  made  direct  from  nodule  tissue  or  crushed 
infusions  failed  to  show  growth.  As  with  the  plates,  there  was  noth- 
ing to  suggest  a  causal  agent,  either  like  or  unlike  Ps.  radicicola.  Little 
reliance  was  placed  on  the  liquid  cultures ;  most  of  them  showed  no 
growth. 

Nitrogen-Fixation  by  Ceanothus  americanus. — In  order  to  test 
the  fixation  of  nitrogen  by  Ceanofhus,  thirteen  young  plants  were 
washed  clean  and  planted  in  clean  quartz  sand  to  which  lime  had  been 
added.  Seven  of  the  plants  were  given  a  nutrient  solution  without 
nitrogen,  and  inoculated  abundantly  with  an  infusion  of  crushed 
nodules.  Six  were  given  the  same  solution  plus  nitrogen,  but  were 
not  inoculated.  None  of  the  plants  fully  recovered  or  made  very  vig- 
orous growth.  After  ten  months  all  those  not  receiving  nitrogen  were 
dead.  Two  of  those  receiving  nitrogen  still  survived  but  were  not 
doing  well.  All  the  plants  produced  nodules. 

Seeds  of  Ceanothus  americanus  were  obtained  in  the  fall  of  1915. 
By  immersing  them  in  commercial  sulfuric  acid  for  ten  minutes  a 
germination  of  about  five  percent  was  obtained.  Three  series  of  six 
pots  were  then  filled  with  white  quartz  sand  and  four  seedlings  planted 
in  each  pot.  Nutrient  solutions  were  added  as  shown  in  Table  10.  The 
plants  in  Series  III  were  inoculated  with  an  infusion  of  crushed 
Ccanottius  nodule. 

After  seven  months,  Series  II  and  III  had  made  a  very  weak 
growth ;  there  Avas  no  choice  between  them.  Series  I  also  had  not  made 
a  very  vigorous  growth,  but  the  plants  were  noticeably  larger  and 
greener  than  those  of  the  other  series.  A  further  observation  of  im- 


PLATE  XV 

Fig.  1. — Longitudinal  section  of  a  Ceanothus  amcriccmus  nodule,  showing 
parasitized  zone.  Stained  with  Flemming's  triple  stain  and  mounted  in  iodine  to 
show  the  starch  X  100 

Fig.  2. — Cross-section  thru  a  similar  nodule  of  Ceanothus  americanus,  showing 
central  cylinder,  some  parasitized  cells,  starch,  etc.  X  100 


X 


•CD 

£ 


™ 


03     O 

^  s 


1917}    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        147 

portance  was  that  Series  III  had  no  nodules,  in  spite  of  the  fact  that 
the  young  seedlings  had  been  abundantly  inoculated  with  an  infusion 
cf  crushed  nodules. 

TABLE  10. — EXPERIMENT  IN  NITROGEN-FIXATION  WITH  SEEDLINGS  OF  CEANOTHUS 

AMERICANUS 


Series 

No.  of 

plants 

Inoculation 

Treatment 

Nodules 

I 
II 
III 

24 

24 
24 

None 
None 
Infusion  of 
crushed  Cean- 
otTius nodule 

Full  nutrient  solution 
Nutrient  solution  without  nitrogen 

Nutrient  solution  without  nitrogen 

None 
None 

None 

While  the  evidence  submitted  is  not  conclusive,  it  at  least  throws 
doubt  upon  the  ability  of  CeanotTius  to  fix  atmospheric  nitrogen.  Ap- 
parently either  quartz  sand  is  not  favorable  or  the  solutions  used  were 
not  best  suited  to  this  plant.  It  must  be  considered,  however,  that 
Ceanotlius  is  a  slow-growing  shrub,  and  hence  the  demonstration  would 
not  be  so  easy  as  with  quick-growing  legumes. 

Histology  of  the  Nodules  of  Ceanotlius  americanus. — An  exten- 
sive description  of  the  nodules  of  Ceanotlius  americanus  is  not  intended 
here,  but  it  is  desired  to  show  enough  of  the  structure  so  that  a  fail- 
comparison  with  legume  nodules  may  be  made.  For  further  informa- 
tion Bottomley,  Spratt,  and  especially  Arzberger,  should  be  consulted. 

In  Plate  XIV  are  shown  some  CeanotTius  nodules.  When  extremely 
young,  they  are  white  or  nearly  so,  and  are  round  or  slightly  oval. 
They  grow  mostly  along  the  longer  axis,  and  become  distinctly  club- 
shaped,  lacking  entirely  the  plumpness  of  legume  nodules.  Branching 
commonly  occurs ;  one  nodule  divides  to  form  two  or  three,  and  later 
these  branches  divide,  the  whole  ultimately  forming  a  cluster  of  con- 
siderable size.  The  structures  are  perennial,  making  new  growth  and 
sending  out  new  branches  each  year.  In  appearance,  as  well  as  to  the 
section  knife,  they  are  quite  woody,  a  point  that  further  distinguishes 
them  from  legume  nodules. 

The  parasitized  zone  of  a  Ceanotlius  nodule  may  be  seen  in 
Plate  XV,  Fig.  1.  The  section  was  mounted  in  iodine  solution,  as 
before  described,  to  show  the  starch.  The  meristem  and  central  cyl- 
inder do  not  show.  Fig.  2  of  the  same  plate  shows  a  cross-section  thru 
a. similar  nodule.  The  central  cylinder  is  well  developed  and  possesses 
a  well  defined  endodermis.  In  the  nodule  cortex  surrounding  the  en- 
dodermis,  there  are  first  several  layers  of  small,  apparently  vacant 
cells,  and  then  a  zone  of  parasitized  cells,  which  are  rather  loosely 
scattered  in  this  area.  The  accumulation  of  starch  in  the  cells  sur- 
rounding the  parasitized  zone  shows  clearly.  As  in  the  legume  nodule, 
there  is  no  epidermis  and  no  root-cap,  but  there  is  a  protective  layer 
of  corky  cells. 


148  BULLETIN  No.  202  [July, 

Plate  XVI  shows  some  of  the  parasitized  cells.  Fig.  1  is  magnified 
430  diameters,  and  Figs.  2  and  3,  1,080  diameters.  The  dark  bodies 
are  said  by  Arzberger  to  be  sporangia  of  a  fungus,  the  hyphae  of  which 
may  be  seen  within  the  cells  at  certain  stages.  He  designates  the 
fnngus  as  Frankia  ceanofhi  Atkinson.  While  not  agreeing  with  Arz- 
berger in  all  the  details,  the  writers  accept  the  fungous1  conceptions  as 
the  true  ones.  The  parasitized  cells  are  tough  and  horny  in  consis- 
tency. When  nodules  were  crushed  for  plating,  these  cells  remained 
intact  and  were  frequently  seen  distributed  thruout  the  agar  plates, 
where  they  were  at  first  mistaken  for  colonies.  No  growth  was  pro- 
duced by  them,  however.  These  parasitized  cells  clearly  bore  no 
resemblance  to  the  bacteroidal  cells  of  the  legumes. 

Summarizing,  the  nodules  of  Ceanofhus  are  unlike  those  of  the 
Leguminosae  in  the  following  points : 

1.  The  CeanotJius  nodules  differ  in  external  appearance  from 
legume  nodules ;  also,  they  are  quite  woody. 

2.  They  are  perennial  structures,  making  new  growth  and  pro- 
ducing new  branches  each  year.    The  mode  of  branching  is  different 
from  that  of  legume  nodules. 

3.  They  contain  a  well  developed  central  cylinder,  resembling 
in  this  respect  a  lateral  root,  of  which  they  may  be  considered  as  a 
modification. 

4.  The  parasitized  cells  are  not  closely  packed  as  in  the  case  of 
legume  nodules,  the  characteristic  bacteroids  of  legume  nodules  are  not 
present,  and  the  cells  do  not  develop  a  central  vacuole  as  do  bacteroidal 
cells.    Instead,  the  parasitized  cells  bear  every  indication  of  containing 
fungous  hyphae. 

5.  No  infection  threads  were  found  in  the  nodules  of  CeanotJius. 

CYCAS  REVOLUTA 

Attempts  to  Isolate  the  Causal  Organism. — Repeated  attempts 
were  made  to  isolate  the  causal  organism  from  nodules  of  Cycas  revo- 
luta  obtained  from  a  greenhouse  plant.  The  results  were  not  wholly 
without  success.  The  nodules  of  Cycas  differ  from  those  of  the  other 
five  groups  of  non-legumes  producing  nodules  in  that  the  older  ones 
become  infected  with  a  blue-green  alga,*  undoubtedly  a  secondary 
infection,  which  renders  the  nodule  less  solid  and  compact.  In  a  cross- 
section  cut  from  one  of  these  older  nodules  the  algal  zone  can  easily 
be  seen  with  the  unaided  eye. 

From  several  algal-infected  nodules  three  forms  of  bacteria  wer-e 
isolated,  none  of  which  resembled  Ps.  radicicola.  Two  were  small, 
deeply  staining  rods,  and  the  third  was  a  larger  rod.  These  three 
organisms  were  tried  in  sand  pot  cultures  upon  Pisum  arvense,  Vicia 
villosa,  Trifolium  pratense,  Medicago  sativa,  Melilotus  alba,  Pliaseolus 

"See  Spratt  w  M;  also  Life.47 


1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        149 

vulgaris,  Vigna  sinensis,  Qlycine  hispida,  Lupinus  perennis,  Arachis 
liypogoea,  Trigonella  foenum-graecum,  Desmodium  canescens,  Amphi- 
carpa  monoica,  Ornithopus  sativus,  and  Onobrychis  sativa.  Only  one 
plant  produced  nodules — Trigonella,  on  which  three  appeared.  This 
fact,  however,  is  not  regarded  as  significant,  since  Melilotus  and 
Medicago  were  without  nodules.  ( The  nodules  of  Trigonella,  Melilotus, 
and  Medicago,  it  has  been  shown,  are  produced  by  the  same  organism.) 
Nodules  due  to  chance  inoculation  are  to  be  expected  when  open  pots 
are  used.  It  was  further  observed  that  the  roots  of  most  of  the  plants 
were  distinctly  brown  and  unhealthy,  as  tho  attacked  by  a  brown  rot. 
The  roots  of  Pisum,  Vicia,  PJiaseolus,  and  Lupinus  seemed  especially 
to  be  injured.  A  small  piece  of  an  unhealthy  lupine  root  was  teased 
apart  and  examined,  disclosing  a  host  of  motile  bacteria. 

Young  Cycas  nodules  in  which  the  algal  zone  was  not  present  were 
plated,  but  without  success.  This  seemed  to  indicate  that  the  organ- 
isms first  isolated  were  not  causal  agents,  but  that  they  followed  the 
alga.  Examination  of  the  algal  infected  nodules  disclosed  a  very  loose, 
open  structure;  indeed,  the  whole  nodule  lacks  the  compactness  of  a 
Ceanothus  nodule.  The  chance  for  entrance  by  the  alga  and  later  by 
foreign  bacteria  is  very  great.  It  would  be  surprising  indeed  if  bac- 
teria were  not  found  in  these  older  nodules. 

ALNUS,  ELAEAGNUS,  AND  MYRICA 

Spratt,52  after  pointing  to  the  demonstration  by  Hiltner45  of  the 
fixation  of  nitrogen  by  Alnus  and  Elaeagnus,  reported  the  isolation  of 
Ps.  radicicola  from  the  nodules  of  these  plants:  The  results  reported 
in  connection  with  the  experiments  are  subject  to  the  following 
criticisms : 

First :  The  demonstration  by  Hiltner  of  the  fixation  of  nitrogen 
by  Alnus  and  Elaeagnus  is  not  nearly  so  convincing  as  Hellreigel  's  and 
Wilfarth's10  discovery  of  the  symbiosis  between  Ps.  radicicola  and 
legumes. 

Second:  Spratt 's  method  of  isolation  is  at  fault.  Spratt  steri- 
lized nodules  and  dropped  them  into  flasks  containing  a  liquid  medium, 
incubating  them  two  days.  Obviously  a  single  foreign  organism  in 
two  days  becomes  a  multitude,  and  no  doubt  a  pure  culture.  It  is  not 
clear  whether  her  agar  plates  were  made  from  fresh  nodule  infusions 
or  incubated  material  as  described  above,  tho  it  appears  that  the  latter 
was  the  case.  The  method  is  unsafe  unless  carried  out  on  a  very 
extensive  scale  (and  then  it  is  questionable),  but  Spratt  used  but  one 
culture  flask  for  Alnus  and  one  for  Elaeagnus,  leaving  one  check, 
which  may  as  well  have  been  omitted.  (Agar  plates  poured  from  legume 
nodules  as  before  described,  seldom  fail  to  give  good  plates  in  this 
laboratory.  If  Ps.  radicicola  is  present  in  Alnus  and  Elaeagnus,  this 
method  should  easily  demonstrate  it.) 


150  BULLETIN  No.  202  [July, 

Third:  The  Kiskalt  amyl-gram  stain  described  by  Harrison  and 
Barlow  and  used  by  Spratt  does  not  identify  Ps.  radicicola.  Numerous 
gram-negative  soil  organisms  lose  the  stain  (aniline- gentian- violet)  in 
ethyl  alcohol,  but  retain  it  when  amyl  alcohol  is  used. 

Fourth:  The  coccoid  form  described  by  Spratt  is  not  analogous 
to  the  bacteroids  of  Ps.  radicicola.  There  is  no  evidence  to  show  that 
Ps.  radicicola  ever  assumes  the  shape  or  characteristics  described  by 
Spratt.  The  writers  have  never  observed  it.  The  only  form  of  Ps. 
radicicola  approaching  a  coccus  form  is  the  extremely  small,  oval 
schwarmer  of  Beyerinck.  The  form  described  by  Spratt  is  entirely 
too  large  for  the  schwarmer. 

Fifth :  The  fixation*  of  nitrogen  in  culture  solutions  is  neither  a 
test  for  Ps.  radicicola  nor  a  proof  of  symbiosis.  With  the  legume 
organism,  the  fixation  of  nitrogen  in  culture  solutions  is  not  significant 
when  compared  with  fixation  in  the  nodule.  Besides,  many  soil  organ- 
isms have  been  attributed  this  power  of  fixation  in  nutrient  solutions. 

Attempts  to  Isolate  the  Causal  Organism. — Nodules  from  Alnus 
glutinosa  and  Myrica  gale  were  obtained  and  plated  out.  The  results 
were  negative.  However,  the  trials  were  not  extensive  because  the 
available  material  was  limited.  Ash  agar,  Spratt 's  agar,  and  beef- 
broth  agar  were  used  as  media. 

CONCLUSIONS 

From  the  foregoing  discussion,  the  following  conclusions  are 
drawn: 

1.  The  root-nodules  of  Ceanoihus,  Cycas,  Alnus,  and  Myrica  are 
not  caused  by  Ps.  radicicola. 

2.  It  is  conceivable  that  Ps.  radicicola  might  enter  the  nodules  of 
Cycas  as  a  secondary  infection  and  function  symbiotically,  but  its 
presence  was  not  demonstrated. 

3.  The  evidence  that  Elaeagnus  and  Podocarpus  nodules   are 
caused  by  Ps.  radicicola  is  not  conclusive. 

4.  Proof  that  these  six  groups  of  plants  are  concerned  with  the 
fixation  of  atmospheric  nitrogen  is  wanting. 


1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        151 

Part  V.— ATTEMPTS  TO  DEVELOP  A  SYMBIOSIS  BETWEEN 
LEGUME  BACTERIA  AND  NON-LEGUME  PLANTS 

Previous  Attempts. — In  1893,  Schneider,18  at  the  Illinois  Experi- 
ment Station,  under  the  direction  of  the  senior  author,  cultivated 
nodule  bacteria  from  Pliaseolus  vulgaris  upon  bean-extract  agar,  then 
upon  a  mixture  of  bean-extract  and  corn-extract  agar,  and  finally  upon 
pure  corn-root-extract  agar.  Transfers  were  made  every  sixth  day. 
After  the  cultures  had  grown  for  a  month  upon  the  pure  corn-root 
extract,  they  were  applied  upon  germinating  seeds  of  corn  and  oats. 
Tho  the  inoculated  corn  plants  produced  no  nodules,  Schneider  claimed 
that  they  were  more  thrifty  than  the  uninoculated  plants.  He  de- 
scribed and  figured  the  infection  of  some  of  the  root-hair  cells,  as 
well  as  some  of  the  epidermal  and  parenchymal  cells.  No  effect  was 
noted  upon  oats. 

That  the  senior  author  was  intensely  interested  in  this  problem 
of  developing  a  symbiosis  between  legume  bacteria  and  non-legume 
plants  is  shown  by  the  fact  that  when  the  opportunity  presented  itself 
after  over  twenty  years,  he  took  up  the  problem  where  Schneider* 
had  left  it. 

Other  attempts  in  this  direction  have  been  reported.  Stutzer, 
Burri,  and  Maul19  inoculated  mustard  plants  with  nodule  bacteria 
which  had  gradually  become  accustomed  to  a  mustard-plant  medium, 
but  without  success.  Grosbiisch31  experimented  with  Graminae,  but 
his  results  were  negative. 

Lemmermann27  studied  the  difference  in  nutrition  between  the 
Lcguminosae  and  the  Graminae.  He  believed  that  the  reasons  for  the 
existence  of  bacterial  symbiosis  in  the  Leguminosae  and  not  in  the 
Graminae  are,  namely,  the  smaller  transpiration  current,  the  higher 
acidity  of  the  root  sap,  and  the  greater  root  development  of  the  former 
as  compared  with  the  latter. 

Preliminary  Discussion. — How  long  ago  symbiosis  between  the 
Leguminosae  and  the  nodule  bacteria  began  cannot  be  estimated.  The 
conditions  under  which  the  first  infection  took  place  are  but  a  matter 
of  conjecture.  Only  a  mass  of  contradictory  literature  concerning  this 
symbiosis  existed  as  a  basis  in  attempting  to  develop  a  symbiosis  be- 
tween these  bacteria  and  non-legume  plants.  There  seemed,  however, 


•The  following  excerpts  are  quoted  from  a  footnote  by  the  senior  author 
introducing  Schneider 's  work  in  1893. 

"Can  the  organisms  be  made  to  grow  upon  these  roots  (grasses  or  cereals) 
by  artificial  means?  . 

' '  It  must  be  confessed  that  it  would  have  been  exceedingly  hazardous  for  any 
one  to  have  expressed  an  affirmative  opinion  upon  this  question;  but  the  vast 
importance  of  the  matter  made  it  desirable  to  try  anything  which  gave  the  least 

promise  of  success While  little  direct  evidence  has  been  gained  in 

favor  of  ultimate  success,  it  is  desirable  to  publish  an  account  of  the  work  so  far 
done,  with  the  hope  of  being  able  at  some  future  time  to  add  greatly  to  the  infor- 
mation now  obtained. ' ' 


152  BULLETIN  No.  202  [July, 

to  be  several  points  of  attack  which  offered  possibilities.  There  was 
some  hope  that  the  organism  might  be  modified  or  changed.  By 
accustbming.it  to  media  containing  juices  of  non-legume  plants,  it 
was  thought  that  it  might  become  so  modified  that  it  would  infect  such 
plants.  Injury  to  the  plant,  especially  nitrogen  starvation,  was  a 
possibility.  Mechanical  injury,  however,  seemed  useless,  since  if  it 
would  develop  a  symbiosis  the  cultivation  of  crops  would  long  since 
have  accomplished  it.  The  non-legume  plants  bearing  nodules,  such  as 
CcanotJius,  Elaeagnus,  and  Cycas,  offered  another  possibility.  It  was 
hoped  that  if  Ps.  radicicola  was  present  in  the  nodules,  the  organism 
would  be  more  adaptable  to  other  non-legume  plants.  In  addition,  the 
fact  that  the  symbiosis  appeared  not  to>  be  confined  to  the  Leguminosae 
alone  was  a  great  encouragement.  It  became  evident,  however,  on 
investigating  nodules  of  non-legumes  that  Ps.  radicicola  was  not 
the  causal  organism;  and  furthermore,  it  seemed  very  doubtful  if 
these  plants  were  concerned  with  fixation  of  atmospheric  nitrogen. 

Another  plan  was  to  obtain  a  non-legume  plant  standing  in  close 
botanical  relationship  to  the  legumes,  and  attempt  to  inoculate  it  with 
legume  bacteria.  The  organism  from  cowpea  nodules  offered  the  greatest 
possibilities,  since  it  seemed  less  particular  in  its  selection  of  a  host  plant. 


Moore,28  in  1905,  reported  that  by  inoculating  legumes  with  nodule 
bacteria  from  Pisum  sativum  which  had  been  grown  for  two  weeks 
upon  nitrogen-free  media,  he  was  able  to  produce  nodules  upon  many 
genera.  He  stated  that  this  was  but  a  single  demonstration  of  numer- 
ous successful  cross-inoculations.  It  appears  from  his  work  that  it 
was  necessary  only  to  grow  the  organisms  upon  nitrogen-free  media 
in  order  to  break  the  special  adaptations. 

Nobbe  and  Hiltner,24  in  1900,  claimed  that  they  were  able  to  make 
the  nodule  bacteria  from  peas  produce  nodules  upon  the  roots  of  beans, 
and  vice  versa. 

Laurent,14  Maze,21  and  Kellerman,  37  among  others,  have  reported 
similar  successful  cross-inoculations. 

EXPERIMENT   I:       COMPARISON    OF    NITROGEN    AND   NITROGEN-FREE   MEDIA 
FOR    THE   GROWTH    OF  PS.   RADICICOLA 

In  order  to  compare  nitrogen  and  nitrogeii-fvec  media  for  the 
growth  of  Ps.  radicicola,  bacteria  from  Melilotus  alba  and  Trifolium 
pratense  were  transferred  to  Freudenreich  flasks  containing  standard 
beef -broth  agar  (No.  205)  and  Fred's  synthetic  agar  (No.  201). 
The  cultures  were  kept  in  the  incubator  at  room  temperature  for 
thirty  months  without  transfer.  Duplicate  cultures  were  transferred 
to  test-tube  slants  once  a  month.  At  the"  end  of  the  thirty  months 
all  cultures  were  transferred  to  ash-agar  slants  for  comparison.  All 


1917}    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        153 

were  alive  and  capable  of  producing  nodules  upon  plants  in  test-tube 
cultures  after  Garman's  method.  Furthermore,  it  is  important  to  note 
that  the  cultures  retained  their  special  adaptation  to  the  original  liost 
plant  and  their  cultural  individualities  as  described  on  pages  136  and 
139. 

EXPERIMENT  II:      COMPARISON   OF  ASH  AGAR  AND  BEEF-BROTH   AGAR   FOR 
THE   GROWTH   OF   PS.    RADICICOLA 

Ash  agar  and  beef-broth  agar  were  compared  as  in  Experiment  I. 
Cultures  of  Trifolium  pratense,  Melilotus  alba,  Vigna  sinensis,  Gly- 
cine  Tiispida,  Robinia  pseudo-acacia,  and  Arachis  hypogoea  were 
transferred  to  Freudenreich  flasks  containing  ash  agar  and  beef -broth 
agar.  Duplicate  test-tube  cultures  transferred  once  a  month  were 
kept.  The  culture  of  Robinia  pseudo-acacia  upon  beef -broth  agar  was 
lost  because  of  a  mold.  After  seventeen  months  the  cultures  were 
transferred  to  ash-agar  slants  for  comparison.  All  were  alive  (except 
the  Robinia)  and  all  retained  their  individual  habit  of  growth. 
Trifolium  pratense  and  Melilotus  alba  were  tested  by  Garman's  method 
and  the  others  in  sand  pot  cultures.  (The  remaining  Robinia  cul- 
tures were  not  tested  except  for  growth.)  All  those  tested  were 
capable  of  producing  nodules  upon  their  original  hosts.  A  few  cross- 
inoculations  were  tried  but  failed.  No  difference  in  virulence  was 
noted. 

EXPERIMENT  III:      GROWTH  OF  PS.  RADICICOLA   ON   TOMATO-STEM   SLANTS 

For  the  purpose  of  infecting  tomato  plants,  cultures  of  Melilotus 
alba  and  Trifolium  pratense  were  grown  upon  tomato-stem  slants  (No. 
421)  placed  in  tubes  containing  standard  beef  broth.  Transfers  were 
made  once  a  month.  After  several  months,  distilled  water  was  substi- 
tuted for  the  broth.  At  the  end  of  twenty-three  months  the  cultures 
were  transferred  to  ash-agar  slants.  Eight  cultures  of  Melilotus  alba 
and  two  of  Trifolium  pratense  were  examined.  All  grew  readily  when 
transferred  to  ash-agar  slants.  Tested  in  agar-tube  cultures,  all  pro- 
duced nodules  upon  their  original  hosts.  Melilotus  alba  bacteria,  how- 
ever, failed  to  inoculate  Trifolium  pratense  plants,  and  Trifolium 
bacteria  failed  with  Melilotus  seedlings. 

EXPERIMENT  IV:      COMPARISON  OF  ASH  AGAR  AND  CONN'S  ASPARAGINATE 

AGAR  AND  THE  EFFECT  OF  SUNLIGHT  ON  GROWTH  AND 

VIRULENCE    OF    PS.    RADICICOLA 

This  experiment  was  designed  not  only  to  compare  nitrogenous 
and  non-nitrogenous  media,  but  also  to  note  the  effect  upon  growth 
and  virulence  of  exposure  to  direct  sunlight.  'Cultures  of  organisms 
from  various  legumes  were  transferred  to  Freudenreich  flasks  of  25-cc. 


154 


BULLETIN  No.  202 


[Julii, 


capacity,  two  sets  containing  Conn's  asparaginate  agar  and  two  ash 
agar.  All  cultures  were  incubated  for  three  days  at  room  tempera- 
ture, after  which  one  set  of  the  asparaginate-agar  flasks  and  one  of  the 
ash-agar  were  removed  to  the  greenhouse  and  left  exposed  to  the  sun- 
light. The  slopes  were  turned  toward  the  south  to  give  maximum 
exposure.  The  checks,  fewer  in  number,  were  left  in  the  incubator. 
The  experiment  covered  three  months — from  March  2  to  June  2,  1916. 
The  temperature  in  the  greenhouse  varied  from  18°  to  42.5°  C.°  The 
highest  temperature  recorded  within  a  similarly  prepared  flask  was 
47°  C.  (thermometer  shaded).  In  Table  11  is  shown  the  arrangement 
of  the  experiment. 

TABLE  11. — ARRANGEMENT  OF  CULTURES  TESTED  ON  ASH  AGAR  AND  CONN  's  ASPARA- 
GINATE AGAR  IN  SUNLIGHT  AND  IN  DARKNESS  :    EXPERIMENT  IV 


Source  of  organism 

In  sunlight 

In  darkness 

Common  name 

Botanical  name 

Ash   agar    |  Conn's  agar 

Ash   agar    [  Conn's  agar 

FlasJc  No. 

Flask  No. 

Flask  No. 

FlasJc  No. 

Acacia 

Acacia  melano- 

Tick  trefoil 

xylon 
Desmodium  canes- 

6011   (died) 

6026 





cens 

6012 

6027 

• 

•  .  •  • 

Dyer's  green- 
weed 

Genista  tinctoria 

6013 

6028 

Soybean 
Sweet  pea 
White  sweet 

Glycine  hispida 
Lathyrus  odoratus 

6014 
6015 

6029 
6030 

6016 

603i 

clover 
Bean 
Trailing  wild 
bean 

Melilotus  alba 
Phaseolus  vulgaris 
Strophostyles 
helvola 

6017   (lost) 
6019 

6020 

6032 
6034 

6035 

6018 

6033 

Red  clover 
Broad  bean 
Cowpea 

Trifolium  pratense 
Vicia  faba 
Vigna  sinensis 

6021 
6023 
6024  (died) 

6036 
6038 
6039   (died) 

6022 
6025 

6037 
6040 

At  the  end  of  three  months  all  cultures  were  transferred  to  ash- 
agar  slants.  Flask  No.  6017  had  been  broken;  the  cultures  in  Nos. 
6011,  6024,  and  6039  had  died.  Of  the  surviving  cultures  those  which 
had  been  kept  in  darkness  recovered  the  most  quickly.  It  was  also 
noted  that  the  organisms  grown  upon  Conn's  asparaginate  agar  were 
the  most  vigorous.  The  ash-agar  cultures  which  had  been  exposed 
to  sunlight  were  the  slowest  in  recovery.  After  several  transfers  upon 
ash  agar,  the  cultures  resumed  their  normal  appearance.  They  were 
then  tested  out  for  virulence  (except  Phaseolus  and  Stroplwstyles) , 
and  it  was  found  that  the  ability  to  produce  nodules  had  not  been 
affected.  The  cultures  of  Melilotus  and  Trifolium  were  tested  by  Gar- 
man's  method  for  ability  to  cross-inoculate  seedlings  of  Trifolium  and 
Melilotus  respectively,  but  the  cultures  were  virulent  only  upon  the 
original  host. 


•The  spring  was  cool  and  cloudy  for  the  most  part,  tho  there  were  some  clear, 
hot  days. 


1917]    POSSIBLE  SYMBIOSIS  SETWEEN  LEGUME  SACTERIA  AND  NON-LEGUMES        iS5 

EXPERIMENTS  ATTEMPTING  THE  INFECTION  OF  NON-LEGUME  PLANTS 
WITH  PS.  RADICICOLA 

The  following  experiments  attempting  the  infection  of  non-legume 
plants  with  Ps.  radicicola  were  but  preliminary.  In  examining  inocu- 
lated plants,  attention  was  given  only  to  any  unexplained  vigor  and 
to  the  presence  or  absence  of  abnormal  root  conditions.  Histological 
technic  was  not  employed. 


EXPERIMENT    V: 


ATTEMPTED    INFECTION    OF    TOMATO    SEEDLINGS    WITH 
SWEET-CLOVER  BACTERIA 


Very  young  tomato  seedlings  were  transferred  from  flats  of  soil 
to  one-gallon  pots  of  limed  white  quartz  sand.  There  were  in  all  one 
hundred  and  fifty  plants.  Half  were  given  a  full  nutrient  solution,*  and 
half  were  given  a  similar  solution  but  without  the  nitrogen.  Copious  in- 
oculations were  made  frequently  with  bacteria  from  sweet  clover  which 
had  been  grown  for  three  weeks,  with  frequent  transfers,  upon  a  decoc- 
tion of  whole  tomato  plants  plus  two  percent  cane  sugar  and  one  per- 
cent peptone  (Medium  No.  111).  After  one  month  the  plants  were 
carefully  washed  free  from  sand  and  examined.  Those  which  had 
been  receiving  nitrogen  were  decidedly  more  thrifty  than  the  others. 
No  abnormal  conditions  were  observed  in  the  roots. 


EXPERIMENT    VI:      ATTEMPTED    INFECTION    OF    TOMATO    SEEDLINGS    WITH 
SWEET-CLOVER  BACTERIA  IN  THE  PRESENCE  OF  COPPER  SULFATE 

Tomato  seedlings  which  had  been  grown  in  flats  of  soil  were 
transferred  to  paper  boxes  containing  limed  white  quartz  sand,  one 
plant  to  each  box.  These  boxes  (2"  x  2"  x  4%")  were  arranged  in  a 
wooden  frame,  sixteen  rows  of  sixteen  each,  making  two  hundred  and 
fifty-six  in  all.  Nutrient  solutions  made  up  as  before  were  used,  except 
that  the  nitrogen  was  varied  as  indicated  in  Table  12.  Inoculations 
were  made  at  the  time  of  transplanting  and  again  after  two  weeks  with 

TABLE  12. — TREATMENT  APPLIED  TO  TOMATO  SEEDLINGS:     EXPERIMENT  VI 


Section  No.  of 
No.     (plants 

Nitrogen  treatment 

Copper-sulfate  treatment 

738 

32 

Full    nutrient    solution*    (10    cc. 

stock  solution  per  liter  water) 

None 

739 

32 

Full  nutrient  solution 

50  cc.  of  1:2500  solution 

740 

32 

Full  nutrient  solution 

50  cc.  of  1:1000  solution 

741 

32 

Full  nutrient  solution 

50  cc.  of  1:500  solution 

742 

32 

Nutrient  solution  without  nitrogen 

None 

743 

32 

Double  nutrient  solution 

None 

744 

32 

Nutrient  solution  without  nitrogen 

50  cc.  of  1:1000  solution 

745 

32 

Double  nutrient  solution 

50  cc.  of  1:1000  solution 

"See  footnote,  page  125. 


156 


BULLETIN  No.  202 


[July, 


bacteria  from  sweet  clover  which  had  grown  upon  tomato-infusion 
peptone  (No.  111).  After  ten  days  copper  sulfate  was  applied  to  the 
sections  indicated  in  amounts  intended  to  stimulate  growth,  to  just 
hinder  growth,  and  to  seriously  retard  growth.  The  treatment  is 
shown  in  Table  12. 

The  plants  were  examined  after  four  weeks.  Those  which  had 
received  the  normal  amount  of  nitrogen  showed  the  best  development. 
The  1 :  2500  solution  of  copper  sulfate  stimulated  both  root  and  top 
development ;  the  1 : 1000  solution  was  slightly  injurious ;  and  the 
1 :  500  damaged  the  plants  seriously.  No  abnormal  conditions  of  the 
roots  were  observed,  except  where  the  1 :  500  copper-sulfate  solution 
was  applied,  in  Avhich  cases  the  injury  was  apparent. 


EXPERIMENT    VII:      ATTEMPTED    INFECTION    OF    TOMATO    SEEDLINGS   WITH 

SWEET-CLOVER  BACTERIA  IN  SOIL  AND  IN  SAND  WITH  VARIED 

NITROGEN  TREATMENT 

Tomato  seedlings  growing  in  sand  and  in  soil  in  an  arrangement 
similar  to  that  of  Experiment  VI  were  inoculated  with  bacteria  from 
sweet  clover  which  had  been  grown  for  ten  months  upon  tomato-stem 
slants  (No.  421) .  Bacteria  were  applied  at  the  beginning  of  the  experi- 
ment and  at  intervals  of  a  week  thereafter.  The  nitrogen  treatments 
used  were  varied  as  shown  in  Table  13. 

TABLE  13. — TREATMENT  APPLIED  TO  TOMATO  SEEDLINGS  IN  SOIL  AND  IN  SAND: 

EXPERIMENT  VII 


Section 

Sand 
or 
'soil 

No.  of 
plants 

Treatment 

Eemarks 

A 

Sand 

256 

Full  nutrient  solution 

B 

Sand 

256 

Nutrient    solution    without 

A  small  amount  of  nitrogen 

nitrogen 

was    added    later,    as    the 

plants  were  starving.    With 

4 

the    exception    of    a    few 

very    weak    plants,    how- 

ever, all  died 

C 

Sand 

224 

Harrison-Barlow     wood-ash 

(No.  100) 

D 

Soil 

288 

Tap  water 

Plants     grew    very    vigor- 

ously and  were  cut  back. 

160     plants     were     given 

1:500  copper  sulfate  solu- 

tion to  further  check  the 

growth 

E 

Sand 

128 

Nutrient   solution   with   Jio 

nitrogen 

F 

Sand 

112 

Nutrient   solution   with   ni- 

trogen trebled 

G 

Soil 

112 

Tap  water 

Plants  were  cut  back 

H 

Soil 

112 

Nutrient   solution  with  ni- 

trogen trebled                       i  Plants  were  cut  back 

1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        157 

In  general  it  may  be  said  that  the  plants  in  soil  were  the.  most 
.vigorous.  Those  in  sand  without  nitrogen  made  very  weak  growth  or 
died.  Those  watered  with  ash  solution  also  made  very  little  growth, 
probably  because  of  a  lack  of  nitrogen.  The  roots  of  the  plants  bore  no 
abnormal  structures. 

EXPERIMENT   VIII:      ATTEMPTED  INFECTION   OF   COMMON   MORNING  GLORY 
'WITH  SWEET-CLOVER  BACTERIA 

The  plan  was  much  like  that  of  Experiment  VII,  except  that  com- 
mon morning  glory  (Convolvulus  major}  was  used.  Seeds  were 
planted  in  sand  and  in  soil,  using  the  paper  boxes  before  described. 
There  were  1,556  plants  in  all.  Inoculations  were  made  at  the  time 
of  planting  and  again  in  two  weeks  with  nodule  bacteria  of  sweet 
clover  which  had  been  grown  for  one  month,  with  frequent  transfers, 
in  an  infusion  of  morning-glory  plants  plus  two  percent  cane  sugar 
and  one  percent  peptone.  Unfortunately  some  of  the  records  were 
lost,  but  Plate  XVII  gives  a  general  idea  of  the  experiment.  The 
inoculation  produced  no  visible  effect.  The  roots  were  carefully  exam- 
ined but  showed  no  unusual  conditions. 

EXPERIMENT   IX:      ATTEMPTED    INFECTION    OF    TOMATO   SEEDLINGS   WITH 

SWEET-CLOVER  BACTERIA  AND  WITH  A  COMPOSITE  INFUSION 

OF  MANY  LEGUME  BACTERIA 

The  experiment  involved  1,268  tomato  seedlings  grown  in  paper 
boxes  filled  with  limed  white  quartz  sand.  These  were  divided  into 
two  equal  sections.  Those  in  one  section  were  given  the  full  nutrient 
solution,  while  those  in  the  other  were  given  the  nutrient  solution 
without  nitrogen.  Half  the  plants  in  each  section  were  inoculated 
with  sweet-clover  bacteria  which  had  been  grown  for  twelve  months 
upon  tomato-stem  slants  (No.  421).  The  other  half  were  inoculated 
with  a  composite  of  all  the  cultures  of  nodule  bacteria  on  hand.  There 
were  cultures  from  forty-five  plant  species,  including  twenty  different 
generic  groups.  Some  were  recent  isolations,  but  most  of  them  had 
been  kept  as  stock  cultures  for  one  to  two  years.  All  had  been  grown 
upon  ash-agar  slants.  The  inoculations  were  in  every  case  without' 
apparent  effect. 

EXPERIMENT  X:      ATTEMPTED  INFECTION    OF   STRAWBERRY  PLANTS   WITH 

SWEET-CLOVER  BACTERIA  AND  WITH  A  COMPOSITE  INFUSION  OF 

BACTERIA  FROM  SEVEN  SPECIES   OF  ACACIA 

Young  strawberry  plants,  one  hundred  and  twenty  in  all,  were 
planted  in  one-gallon  pots  of  sand  and  of  soil  and  treated  as  shown 
in  Table  14.  Half  the  plants  in  each  series  were  inoculated  with  sweet- 


158 


BULLETIN  No.  202 


[July, 


PLATE  XVII 

Experiment  VIII:  Morning-glory  plants  grown  in  sand  and  in  soil  inocu- 
lated with  sweet-clover  bacteria  grown  for  one  month  in  morning-glory  infusion 
media 


1&17]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        159 


TABLE  14. — TREATMENT  APPLIED  TO  STRAWBERRY  PLANTS  IN  SAND  AND  IN  SOIL: 

EXPERIMENT  X 


Series 

No.  of 
plants 

Medium 

Treatment 

A 
B 
C 
D 

30 
30 
30 
30 

Good  potting  soil 
1  part  soil  to  4  parts  sand 
Washed  yellow  sand 
Washed  yellow  sand 

Tap  water  only 
Tap  water  only 
Full  nutrient  solution 
Full  nutrient  solution  without 
nitrogen 

clover  bacteria;  the  other  half  were  inoculated  with  a  composite  in- 
fusion of  bacteria  from  seven  species  of  Acacia^  Heavy  inoculations 
were  made  frequently ;  the  bacteria  used  had  been  grown  upon  ash-agar 
slants. 

The  plants  did  well  at  first,  but  later  became  infested  with  red 
spiders  and  in  spite  of  sprays  did  not  make  a  satisfactory  growth.  The 
results  were  negative. 


160  BULLETIN  fro.  20%  [July, 

SUMMARY 

1.  The  nodule  bacteria  studied  were  found  to  be  true  Sctiizo- 
mycetes,  actively  motile  by  means  of  a  single  polar  flagellum. 

2.  These  bacteria  may  be  divided  into  groups  according  to  the 
host  plants  to  which  they  become  specifically  adapted.    In  addition  to 
the  cross-inoculations  previously  known,  many  new  ones  were  found 
to  exist.    These  are  given  under  Group  III,  page  136. 

3.  In  addition  to  these  special  adaptations,  there  are  among  the 
various  nodule  bacteria  serological  and  cultural  differences  which  are 
permanent,  giving  perhaps  a  legitimate  basis  for  the  belief  that  distinct 
species  exist.    In  numerous  other  characteristics,  however,  the  nodule 
bacteria  are  so  strikingly  alike,  and  as  a  whole  they  differ  so  widely 
from  any  other  species  of  bacteria,  that  it  seems  more  consistent  to 
regard  the  adapted  forms  as  varieties  of  the  single  species  Pseudomo- 
nas  radicicola. 

4.  The  legume  nodule  originates  in  the  root-cortex,  much  as  does 
the  lateral  root,  but  here  the  similarity  ends.     The  nodule  consists 
chiefly  of  a  mass  of  parenchymal  cells  which  are  devastated  by  the 
nodule  bacteria  giving  way  to  the  bacteroid  forms  of  the  invading 
organism,  which  then  make  up  the  greater  part  of  the  cell  contents. 

5.  The  nodules  of  the  non-legumes  Ceanothus,  Cycas,  Alnus,  and 
Myrica,  said  to  be  concerned  with  the  fixation  of  atmospheric  nitrogen, 
are  not  caused  by  Pseudomonas  radicicola.    The  nodules  of  Ceanothus 
are  wholly  different  morphologically  from  those  of  the  Leguminosae. 
The  evidence  that  the  nodules  of  Elaeagnus    and   Podocarpus    are 
caused  by  these  organisms  is  not  conclusive.    Furthermore,  the  proof 
that  any  of  these  six  groups  of  plants  are  concerned  in  the  fixation 
of  atmospheric  nitrogen  is  not  conclusive. 

6.  The  adaptations  of  the  nodule  bacteria  are  constant.     Such 
factors  as  the  use  of  organic  or  inorganic  substances  in  the  medium, 
the  acidity  or  alkalinity  of  the  medium,  and  the  presence  or  absence 
of  combined  nitrogen  in  the  same,  do  not  affect  the  virulence  nor 
break  the  special  adaptations.    The  virulence  and  specificity  are  bound 
up  with  the  life  of  the  organism. 

7.  The  preliminary  experiments  here  reported  attempting  the 
infection  of  non-legume  plants  with  nodule  bacteria  failed. 

8.  No  conclusions  can  be  drawn  as  to  the  possibility  or  probability 
of  developing  or  finding  nodule  bacteria  that  will  grow  on  non-legume 
plants.    The  constancy  of  the  special  adaptations  and  the  fact  that  no 
plants  other  than  legumes  harbor  the  organisms  in  question,  as  had 
been  supposed,  have  been  discouraging  and  to  some  degree  limit  the 
hope  of  ultimate  success. 


1017]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        161 


PART  VI.— BIBLIOGRAPHIES1 

(a)    SYMBIOTIC  NITROGEN  FIXATION  BY  LEGUMES 

1687  MALPIGHI.     Anatomic  plantarum  pars  sec.  de  gallis.     Op.    (1687),  2,  126, 

(Leiden). 
1825  DECANDOLLE.    Memoircs  sur  la  famille  des  L6gumineuses  (1825)j 22.    (Paris). 

1837  BOUSSINGAULT.     Becherches  chimiques  sur  la  vegetation  enterprises  dans  le 

but  d  'examiner  si  les  plantes  prennent  de  1  'azote  de  1  'atmosphere.  Ann. 
Sci.  Nat.  Bot.  (1837),  10,  257. 

1838  BOUSSINGAULT.     Kecherches  chimiques  sur  la  vegetation  enterprises  dans  le 

but  d 'examiner  si  les  plantes  prennent  de  1 'azote  del 'atmosphere.  Compt. 
Eend.  Acad.  Sei.  (Paris)   (1838),  6,  102-112,  and  7,  889-892. 
BOUSSINGAULT.     Eecherches  chimiques  sur  la  vegetation  enterprises  dans  le 
but  d  'examiner  si  les  plantes  prennent  de  1  'azote  de  1  'atmosphere.    Ann. 
Chim.  et  Phys.  (1838),  67,  1-54,  and  69,  353-367. 

1839  BOUSSINGAULT.     Eecherches  chimiques  sur  la  vegetation  enterprises  dans  le 

but  d  'examiner  si  les  plantes  prennent  de  1  'azote  de  1  'atmosphere.  Jour. 
Prakt.  Chem.  (1839),  16,  385.  Compt.  Eend.1  Acad.  Sci.  (Paris)  (1839), 
7,  889. 

1850  VILLE.     Note  sur  1 'assimilation  de  1 'azote  de  1'air  par  les  plantes.     Compt. 

Eend.  Acad.  Sci.  (Paris)   (1850),  31,  578. 

1851  GASPAEINI.     Ovservazioni  sulla  struttura  dei  spongiolari  di  alcune  piante 

Leguminose.  Lette  All.  Ac.  Napoli  (1851).  Cited  by  Tschirch,  Ber.  Deut. 
Bot.  Gesell.  (1887),  5,  81. 

VILLE.  Note  sur  1 'assimilation  de  1 'azote  de  1'air  par  les  plantes.  Jour. 
Prakt.  Chem.  (1851),  52,  60. 

1852  VILLE.     Eecherches  experimentales  sur  la  vegetation.    Abst.  in  Compt.  Eend. 

Acad.  Sci.  (Paris)  (1852),  35,  464-468,  650-654. 

1853  TREVIRANUS.     Ueber    die    Neigung    der    Hiilsengewachse    zu    unterirdischer 

Knollenbildung.    Bot.  Ztg.  (1853),  11,  393. 

1854  BOUSSINGAULT.     Eecherches  sur  la  vegetation.     Compt.  Eend.     Acad.     Sci. 

(Paris)   (1854),  38,  580-607,  and  39,  601-613. 

VILLE.  Absorption  de  1 'azote  de  1'air  par  les  plantes.  Jour.  Prakt.  Chem. 
(1854),  62,  121,  183.  Abst.  in  Compt.  Eend.  Acad.  Sci.  (Paris)  (1854), 
38,  705-709,  723-727. 

1855  HARTING.     Eecherches  concernant  1 'assimilation  de  1 'azote  de  1'air  par  les 

vegetaux.    Compt.  Eend.  Acad.  Sci.  (Paris)   (1885),  41,  942-946. 

1856  VILLE.     De  1  'etat  auquel  se  trouve  quand  il  est  absorbe  1  'azote  que  les  plantes 

tirent  de  1'air.     Compt.  Eend.  Acad.  Sci.   (Paris)    (1856),  43,  143-148. 

1857  BOTJSSINGAULT.     Eecherches  sur  1 'influence  que  1'azote  assimilable  des  engrais 

exerce  sur  la  production  de  la  matiere  vegetale.  Ann.  Sci.  Nat.  Bot. 
(1857),  7,  5.  Compt.  Eend.  Acad.  Sci.  (Paris)  (1857),  44,  940-953. 

1858  LACHMANN.     Ueber  Knollchen  dcr  Leguminosen.     Landw.     Mitt.     Zeit.   K. 

Lehranstalt.  u.  vers.  Stat.  (1858),  37.  Centbl.  Agr.  Chem.  (1891),  20,  837. 
1861  BRETSCHNEIDER.     Kann  der  freie  Stickstoff  zur  Bildung  der  Leguminosen 
beitragen?     Jahresber.  Agr.  Chem.   (1861),  4,  123. 

*In  the  preparation  of  these  bibliographies  assistance  vas  also  rendered  by 
Albert  L.  Whiting,  Associate  in  Soil  Biology,  Warren  E.  Schoonover,  First  Assistant 
in  Soil  Biology,  and  William  A.  Albrecht,  Fellow  in  Agronomy. 


162  BULLETIN  No.  202 

LA  WES  AND  GILBEKT.  On  the  sources  of  nitrogen  vegetation  with  special 
reference  to  the  question  whether  plants  assimilate  free  or  uncombined 
nitrogen.  Phil.  Trans.  Eoy.  Soc.  London  (1861),  151,  431-579.  Abst. 
in  Proc.  Roy.  Soc.  London  (1859-60),  10,  544-557. 

1863  LA  WES  AND  GILBERT.     On  the  assimilation  of  nitrogen.    Chem.  News  (1863), 

7,  127-128. 

1864  RAUTENBERG  UNO  K#HN.     Vegetationsversuche  im   Sommer   1863.     Landw. 

Vers.  Stat.  (1864),  6,  355-359, 

1866  1WoRONiN.    Ueber  die  bei  der  Schwarzerle   (Alnus  glutinosa)   und  bei  der 

gewohnlichen  Gartenlupine  (Lupinus  mutabilis)  auftretenden  Wurzelan- 
schwellungen.  MSm.  Acad.  Imp.  Sci.  (St.  Petersburg)  (1866),  ser.  7, 
10,  No.  6. 

1867  rWoRONiN.     Observations  sur  certaines  excroissances  que  presentant  les  racines 

de  1'aime  et  du  lupin  des  jardins.     Ann.  Sci.  Nat.  Bot.  (1867),  ser  5, 
7,  73. 
1871  DEHERAIN.    Sur  1 'absorption  de  1 'azote  par  les  plantes.     Bui.  Soe.  Chim. 

(1871),   16,    2. 

DEHERAIN.  Sur  1 'intervention  de  1 'azote  atmosphe'rique  dans  la  ve'ge'tation. 
Abst.  in  Compt.  Rend.  Acad.  Sci.  (Paris)  (1871),  73,  1352-1356. 

1873  DEHERAIN.    Sur  1 'intervention  de  1 'azote  atmosphe'rique  dans  la  ve'ge'tation. 

Bui.  Soc.  Chim.  (1873),  19,  538-544. 
DEHERAIN.     Sur  1 'intervention  de  1 'azote  atmosphe'rique  dans  la  ve'ge'tation. 

Compt.  Rend.  Acad.  Sci.  (Paris)  (1873),  76,  1390-1394. 
•ERIKSSON.     Studier  b'fver  Leguminonsernas  rotknolar.  Acta  TJniv.  Lundensis. 

Lunds.     Univ.  Ars-skrift,  IIAfdelningen  for  Math.  och.  Natur.  Lund 

(1873),  10,  No.  8,  1-30.    Ref.  Bot.  Ztg.  (1874),  32,  381-384. 

1874  SESTINI.     Thatigkeitberichte    aus    den    landwirthschaftlichen    Ursuchs-Sta- 

tionen.     Landw.  Vers.  Stat.  (1874),  17,  144-145. 

1878  CORNU.     fitudes    sur    le    Phylloxera    vasatatrix.     Mem.    Acad.    Sei.     Paris 

(1878),  26,  159. 

1879  *FRANK,  B.     Ueber  die  Parasiten  in  den  Wurzelanschwellungen  der  Papi- 

lionaceen.    Bot.  Ztg.  (1879),  37,  377-388,  393-400. 
KNY,    L.     Ueber    die    Wurzelanschwellungen    der    Leguminosen    und    ihre 

Erzeugung  durch  Eir.fluss  von  Parasiten.    Sitzber.  Bot.  Ver.  Prov.  Brand- 
enburg  (1879),  21,  55.     Bot.  Ztg.   (1879),  37,  57. 
KNY,  L.    Zu  dem  Aufsatze  des  Herrn  Prof.  B.  Frank  "Ueber  die  Parasiten 

in  den  Wurzelanschwellungen  der  Papilionaceen. "     Bot.  Ztg.    (1879), 

37,  537-541. 
PRILLIEUX.    Sur  la  nature  et  sur  la  cause  de  la  formation  des  tubercles  qui 

naissent  sur  les  racines  des  Le"gumineuses.     Bui.  Soc.  France   (1879), 

26,  98-106. 

1884  ATWATER.     On  the  assimilation  of  atmospheric  nitrogen  by  plants.     Rpt. 

Brit.  Assoc.  Adv.  Sci.  (1884),  54,  685. 
SCHINDLER.     Zur  Kenntnis   der  Wurzelknollchen   der   Papilionaceen.     Bot. 

Centbl.  (1884),  18,  84-88. 
TROSCHKE.    Ueber  die  Kultur  der  Lupine  in  wasseringen  Nahrlosung  und 

ueber  die  Zusamensetzung  der  Wurzelanschwellungen.     Wochenbl.  Pom- 

mersch.     Okon.  Gesel.  (1884),  125.    Just's  Bot.  Jahresber.     (1884),  12, 

Part.  1,  60. 

1885  ATWATER  AND  WOODS.    The  acquisition  of  atmospheric  nitrogen  by  plants. 

Amer.  Chem.  Jour.  (1885),  6,  365. 
BRUNCHORST.    Ueber   die   Knollchen    and   den   Leguminosenwurzeln.     Ber. 

Deut.    Bot.    Gesell.    (1885),    3,    242-257.      Untersuchungen    Bot.    Inst. 

(Tubingen)   (1886),  2,  Heft.  1,  151. 
SCHINDLER.    Ueber  die  biologische  Bedeutung  der  Wurzelknollchen  bei  den 

Papilionaceen.    Jour.  f.  Landw.  (1885),  33,  325-336. 


1917]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        163 

1886  ATWATER.     On  the  liberation  of  nitrogen  from  its  compounds  and  the  acquisi- 

tion of  atmospheric  nitrogen  by  plants.     Amer.  Chem.  Jour.    (1886), 
8,  398-420. 

FRANK,  B.  Die  Stickstoff-Frage  vor,  auf  und  nach  der  Naturforscher- 
Versammlung  zu  Berlin.  Deut.  Landw.  Presse  (1886),  Nr.  97,  629-630. 
Kef.  in  Jahresber.  Agr.  Chem.  (1886),  29,  91. 

FRANK,  B.  Ueber  die  Quellung  der  Stickstoffnahrung  der  Pflanzen.  Ber. 
Deut.  Bot.  Gesell.  (1886),  4,  293-301. 

GILBERT.  Einige  neue  Ergebnisse  betr.  d.  Stickstoff  Quelle  d.  Pflanze.  Chem. 
Centbl.  (1886),  57,  872. 

HARTIG.     Ueber    die    symbiotische    Erscheinungen    im    Pflanzenleben.     Bot. 

Centbl.  (1886),  25,  350. 
•HELLRIEGEL.    Welche  Stiekstoffquellen  stehen  der  Pflanze  zu  Gebote?  Tagebl. 

59,  Versamml.  Deut.  Naturf.  u.  Aerzte   (Berlin)    (1886),  290.     Landw. 

Vers.  Stat.  (1886),  33,  464-465. 

•PRA^MOWSKI.  Ueber  die  Wurzelknollchen  der  Leguminosen.  Bot.  -Centbl. 
(1886),  36,  215-219,  248-255,  280-285. 

SORAUER.     Wurzelknollen.     Pflanzenkrankheiten.      (1886),    1,    743-748,   and 

2,  73-74. 
STRECKER.     Die  Bereicherung  des  Bodens  durch  den  Anbau  "  berichernder " 

Pflanzen.    Jour.  Landw.  (1886),  34,  1-82. 

1887  BENECKE.    Ueber  die  Knollchen  an  den  Leguminosenwurzeln.    Bot.  Centbl. 

(1887),  29,  53-54. 

BERTHELOT.  Sur  la  fixation  direct  de  1  'azote  gaseux  de  1  'atmosphere  par  les 
terres  v6g6tales  avec  le  concours  de  la  ve'gStation.  Compt.  Rend.  Acad. 
Sei.  (Paris)  (1887),  104,  205-209,  625,  630. 

BEYERINCK.  Wurzelknospen.  Verh.  K.  Akad.  Wetensch.  (Amsterdam) 
(1887),  25. 

HELLRIEGEL.  Ueber  die  Beziehungen  der  Baeterien  zu  der  Stickstoffernahrung 
der  Leguminosen.  Centbl.  f.  Bakt.  1  Abt.  (1887),  1,  133. 

MATTIROLO  E  BUSCAGLIONI.  Si  contengono  bacteri  nei  tubercoli  radical! 
delle  Leguminose?  Malpighia  (1887),  1,  464-473. 

MATTIROLO  E  BUSCAGLIONI.  Ancora  sui  baeteroidi  delle  Leguminose?  Mal- 
pighia (1887),  1,  536-541. 

SORAUER.  Zusamenstellung  der  neuern  Arbeiten  ueber  die  Wurzelknollchen. 
und  deren  als  Bakterien  angesprochene  Inhaltsporchen.  Bot.  Centbl. 
(1887),  31,  308-314.  343-345. 

'TSCHIRCH.     Beitrage  zur  Kenntnis  der  Wurzelknollchen  der  Leguminosen. 

Ber.  Deut.  Bot.  Gesell.  (1887),  5,  58-98.    Centbl.  f.  Bakt.  1  Abt.  (1887), 

1,  634-641. 
TSCHIRCH.    Ueber  die  Wurzelknb'llchen  der  Leguminosen  Wurzeln.     Gesell. 

Naturw.  Freunde  Berlin  (1887),  No.  4.    Bot.  Centbl.  (1887),  31,  224-225. 

TSCHIRCH.  Ueber  die  Entwickelungsgeschichte  der  Bakteroiden  in  der 
Wurzeln  der  Leguminosen.  Tagebl.  60  Versamml.  Deut.  Naturl.  u. 
Aerzte  zu  Wiesbaden,  1887. 

•WARD.  On  the  tubercular  swellings  on  the  roots  of  Vicia  faba.  Phil.  Trans. 
Roy.  Soc.  London  (1887),  ser.  B,  178,  539-563. 

WIGAND.     Bakterien  innerhalb  der  geschlossenen  gewebea  der  knollenartigan 

Ansehwellungen  der  Papilionaceen  Wurzeln.     Bot.  Blefte  Forsch.  Bot. 

Gart.  (Marburg)   (1887),  2,  88-97. 
WILFARTH.     Ueber    Stickstoffaufnahme    der    Pflanzen.      Tageblatt    der    60 

Versamml.  Deut.  Naturf.  u.  Aerzte  zu  Wiesbaden,  1887,    See  also  Landw. 

Vers.  Stat.  (1887),  34,  460. 


164  BULLETIN  No.  202  [July, 

1888  BERTHELOT.     Experiences  nonvelles  sur  la  fixation  de  1  'azote  par  certaines 

terres  vegetales  et  par  certaines  plantes.  Compt.  Kend.  Acad.  Sci.  (Paris) 

(1888),  107,  372-378. 
°BEYERINCK.     Die  Bacterien  der  Papilionaceenknollchen.     Bot.  Ztg.    (1888) 

46,  725-735,  741-750,  758-771,  782-790,  797-803. 
BREAL.     Observations  sur  la  fixation  de  1 'azote  atmospherique  par  lea  Le"- 

gumineuses  dont  les  racines  portent  des  nodosites.     Compt.  Eend.  Acad. 

Sci.  (Paris)   (1888),  107,  397-400. 
BREAL.     Observations  sur  les  tubercles  a  bacteries  qui  se  developpement  sur 

les  racines  des  Legumineuses.     Ann.  Agron.  (1888),  14,  481-495. 
BUCKHOUT.       Experiment  on  the  production  of  root  tubercles.     Penn.  Agr. 

Exp.  Sta.  Ann.  Ept.  (1888),  134-136. 
CHEVREUL."    Sur  le  role  de  1 'azote  atmospherique  dans  1'economie  vege'tale. 

Compt.  Eend.  Acad.  Sci.  (Paris)   (1888),  106,  1460-1461. 
DELPINO.     Osservazioni  topra  i  fcatteriocecidii  e  la  sorgente  d '  azote  in  una 

pianta  de  Galega  officinalis.     Malpighia  (1888),  2,  385-394. 
FARLOW.      Tubercles  on  leguminous  roots.    Gard.  and  Forest  (1888),  1,  135. 
FRANK,  B.     Ueber  den  Einfluss  welchen  das  Sterilisiren  des  Erdbodens  auf 

die  Pflanzen-Entwickelung  ausiibt.     Ber.  Deut.  Bot.  Gesell.    (1888),  6, 

87-97. 
FRANK,  B.     Untersuchungen  ucber  die  Ernahrung  der  Pflanze  mit  Stickstoff 

und   ueber    den   Kreislauf   desselben   in    der   Landwirtschaft.     Landw. 

Jahrb.  (1888),  17,  421-552. 
GAUTIER  ET  DROUIN.     Eecherches  sur  la  fixation  de  1 'azote  par  le  sol  et  les 

vegetaux.      Compt.    Eend.    Acad.    Sci.     (Paris)     (1888),    106,    754-757, 

863-866,  944-947,  1098-1101,  1174-1176,  1232-1235,  1605,1607. 
HELLRIEGEL     UND     WILFARTH.     Ueber     Stickstoffaufnahme     der     Pflanzen. 

Centbl.  Agr.  Chem.  (1888),  17,  228.     Abst.  from  Tagebl.  60  Versamml. 

Deut.  Naturf.  Aerzte  n.  Wiesbaden,  362-363. 
.    "HELLRIEGEL  UND  WILFARTH.     Untersuchungen  ueber  die  Stickstoff-Nahrung 

der  Gramineen  und  Leguminosen.    Beil.  Zeit.  d.  Verins.  fur  die  Euben- 

sucker  Industrie  (Berlin)   (1888),  1-234. 
LUNDSTRoM.     Ueber  Mykodomatien  in  den  Wurzeln  der  Papilionceen.     Bot. 

Centbl.  (1888),  33,  159-160,  185-188. 
PICHI.     Alcune   osservazioni   sui  tubercoli  radical!   delle  Leguminose.     Atti 

Soc.  Toscani  Sci.  Nat.  (1888). 
SCHLOESING.     Sur    les    relationes    de    1 'azote    atmospherique    avec    la    terre 

vegetale.    Eeponse  aux  observations  de  M.  Berthelot  inserts  aux  Compt. 

Eend.  du  9fAvril.  Compt.  Eend.  Acad  Sci.  (Paris)  (1888),  106,  1123-1129. 
SCHLOESING.     Sur   les    relationes    de   1 'azote    atmospherique    avec    la    terre 

vegetale.    Compt.  Eend.  Acad.  Sci.  (Paris)  (1888),  106,  805-809,  898-902, 

982-987,  and  107,  290,  296,  301. 
TIEGHEM  VAN  ET  DouLiOT.     Origine,  structure,  et  nature  morphologique  'de  i 

tubercles  radicaux  des  Legumineuses.     Bui.  Soc.  Bot.  France  (1888),  35, 

105-109. ' 
VINES.     On  the  relation  between  the  formation  of  tubercles  on  the  roots  of 

Leguminosae  and  the  presence  of  nitrogen  in  the  soil.  Ann.  Bot.  (1888-9), 

2,  386-389. 
"VuiLLEMlN1.     Les  tubercles  radicaux  des  Legumineuses.     Ann.  Sci.  Agron. 

(1888),  1,  121-122. 
WARD.     Some  recent  publications  bearing  on  the  question  of  the  sources  of 

nitrogen  in  plants.     Ann.  Bot.   p.888),  1,  325-357. 

1889  ATWATER.     Atmospheric    nitrogen    as   plant    food.      Conn.    Agr.    Exp.    Sta. 

(Storrs)  Bui.  5,  (1889). 


1017]    POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        165 

ATWATER  AND  WOODS.     The  acquisition  of  atmospheric  nitrogen  by  plants. 

Conn.  Agr.  Exp.  Sta.  Ann.  Ept.  (Storrs)   (1889),  11-51. 

BERTHELOT.     Fixation  de  1 'azote  par  la  terre  vegetale,  nue,  ou  avec  le  con- 
course des  Legumineuses.    Compt.  Eend.  Acad.  Sci.  (Paris)   (1889),  108, 

700-708. 
BERTHELOT.     Sur  la  fixation  de  1 'azote  atmospherique.    Compt.  Eend.  Acad. 

Sci.  (Paris)   (1889),  109,  417. 
BERTHELOT.     Experiences  nouvelles  sur  la  fixation  de  1 'azote  par  certaines 

terres  vegetales  et  par  certaines  plantes.    Ann.  Chim.  et  Phys.    (1889), 

316,  433-638. 
BREAL.     Experiences  sur  la  culture  des  Legumineuses.    Ann.  Agron.  (1889), 

15,  529-551. 
BR&AL.     Fixation  de  1 'azote  par  les  Legumineuses.    Compt.  Rend.  Acad.  Sci. 

(Paris)    (1889),  109,  670-673. 
BUCKHOUT.     Experiments  on  the  production  of  root  tubercles.     Penn.  Agr. 

Exp.  Sta.  Ann.  Ept.  (1889),  177-181. 
FRANK,    B.     Ueber    die   Pilzsymbiose    der   Leguminosen.     Ber.    Deut.    Bot. 

Gesell.  (1889),  7,  332-346. 

FRANK,  B.      "Ueber  den  gegenwartigen  Stand  nnserer  Kentniss  der  Assimila- 
tion elementaren  Stickstoffs  durch  die  Pflanze.    Ber.  Deut.  Bot.  Gesell. 

(1889),  7,  234-247. 
HELLRIEGEL.     Bemerkungen  zu   dem  Aufsatze  von  B.  Frank   "Ueber   den 

Einfluss  welchen  das  Sterilisiren  der  Erdbodens  auf  die  Pflanzen-Ent- 

wicklung  ausiibt."    Ber.  Deut.  Bot.  Gesell.    (1889),  7,  131-137. 
HELLRIEGEL  UNO  WILFAETH.     Erfolgt  die  Assimilation  des  freien  Stickstoffs 

durch  die  L6gum;nosen  unter  Mitwirkung  niederer  Organismen?     Ber. 

Deut.  Bot.  Gesell.  (1889),  7,  138-143. 
KELLER.     Die  Wurzelknollchen  der  Leguminosen.     Biol.  Centbl.   (1889),  9, 

97-106. 
LA  WES  AND  GILBERT.     On  the  present  position  of  the  question  of  the  sources 

of  nitrogen  in  vegetation,  with  special  references  to  the  question  whether 

plants  assimilate  free  or  uncombined  nitrogen.     Phil.  Trans.  Eoy.  Soc. 

London  (1889),  180,  1-107.    See  also  Proc.  Eoy.  Soc.  London  (1887-8), 

43,  108-116,  and  (1887),  42,  483. 
LONAY.    La  question  de  1 'azote  et  culture  les  Legumineuses.     (Nivelles) 

(1889),  Just's  Bot.  Jahresber.    (1889),  17,  7. 

12PRA2MOWSKi.     Das  Wesen  und  die  biologische  Bedeuntung  der   Wurzel- 
knollchen der  Erbse.     Bot.  Centbl.  (1889),  39,  356-362. 

SALFELD.     Ueber    die   Verwertung    der    Hellriegel 'schen   Versuche    mit   Le- 
guminosen im  landwirtschaft.     Centbl.  Agr.  Chem.   (1889),  18,  239-244. 
SCHLOESING.     Sur    les    relations    de    1 'azote    atmospherique    avec    la    terre 

vegetale.     Eeponse  a  M.  Berthelot.     Compt.  Eend.  Acad.  Sci.    (Paris) 

(1889),  109,  345-349.. 
SCHROTER.     Phytomyxinae.     Engler  und  Prantl:     Die  Naturlichen  Pflanzen- 

familien,  I  Teil,  1  Abt.,  7. 
VUILLEMIN.     Les  tubercles  des  Legumineuses  et  leurs  habitants.     Bui.  Soc. 

Sc.  Nancy  (1889),  2  ser.  9,  Fasc.  22.    Just's  Bot.  Jahresber.  (1889),  17, 

Part  I,  583. 
"WARD.     On  the  tubercles  of  leguminous  plants,  with  special  reference  to 

the  pea  and  bean.    Proc.  Eoy.  Soc.  London  (1889),  46,  431-443. 
1890  ATWATER  AND  WOODS.     The  acquisition  of  atmospheric  nitrogen  by  plants. 

Conn.  Agr.  Exp.  Sta.  Ann.  Ept.  (Storrs)   (1890),  12-14. 
BERTHELOT.     Observations  sur  les  reactions  entre  la  terre  vegetale  et  1'am- 

moniaque   atmospherique.     Compt.   Eend.   Acad.    Sci.    (Paris)    (1890), 

110,  558-560. 


166  BULLETIN  No.  202  [July, 

BERTHELOT.     Observations   sur   la   note  pre'ce'dente  de  Schloesing,  Fils,  et 

Laurent.    Compt.  Bend.  Acad.  Sci.  (Paris)  (1890),  111,  753. 
BEYERINCK.     Kiinstiche  Infektion  von  Vicia  faba  mit  Bacillus  radicicola: 

Ernahungsbedingungen  dieser  Bacterien.    Bot.  Ztg.  (1890),  43,  837-843. 

Bot.  Centbl.  (1891),  45,  247-248. 
CONN.     Nature  of  the  root  tubercles  of  leguminous   plants.     U.  S.  Dept. 

Agr.  Exp.  Sta.  Bee.  (1890-91),  2,  686,  and  (1891-92),  3,  56-64. 
FRANK,  B.     Ueber  die  Pilzsymbiose  der  Leguminosen.    Landw.  Jahrb.  (1890), 

19,  522-640. 
FRANK,  B.    Ueber  Assimilation  von  Stickstoff  aus  der  Luft  durch  Bobinia 

Pseudoacaeia.    Ber.  Deut.  Bot.  Gesell.  (1890),  8,  292-294. 
FRANK,  B.,  UNO  OTTO.     Untersuchungen  ueber  Stickstoff  Assimilation  in  der 

Pflanze.    Ber.  Deut.  Bot.  Gesell.  (1890),  8,  331-342. 
HELLRIEGEL.     Ueber  Stickstoffnahrung  landwirtsehaftlicher  Kulturgewachse. 

Ber.  Internat.  Land.  Forstwiss.  Congress  zu  Wien  (1890),  Sec.  V.  Subsec. 

b,  80,  15.    Koch's  Jahresber.     (1890),  1,  131. 

KLEBAHN.    Die  neuesten  Untersuchungen  ueber  die  Wurzelknollehen.     (Hum- 
bolt)  (1890),  148. 

KOCH,  A.     Zur  Kenntniss  de  Faden  in  den  Wurzelknollehen  der  Legum- 
inosen.  Bot.  Ztg.  (1890),  48,  607-615. 
LACHMANN.     Ueber  Knollen  an  den  Wnrzeln  der  Leguminosen.    Centbl.  Agr. 

Chem.  (1890),  20,  837-854. 
LAURENT.      Experiences  sur  la  production  de  nodosite's  chez  le  pois  a  la  suite 

d 'inoculations.     Bui.  Acad.  Boy.   Belgique    (1890),  3   ser.,  19,  No.  6, 

764-771. 
"LAURENT.     Sur  le  microbe  des  nodosites  des  Legumineuses.     Compt.  Eend. 

Acad.  Sci.  (Paris)   (1890),  111,  754.    Bot.  Centbl.  (1891),  No.  8. 
LAWES  AND  GILBERT.     New  experiments  on  the  question  of  the  fixation  of 

free  nitrogen.    Proc.  Eoy.  Soc.  London  (1890),  47,  85-118. 
NOBBE.     Ueber  die  Stickstoffernahrung  der  Leguminosen.    Verhandl.  Gesell. 

Deut.  Naturf.  u.  Aerzetezu  Bremen  (1890),  2,  551. 
PETERMANN.     Beitrage  zur  Stickstofffrage;     Chem.  Centbl.    (1890),  ser  4, 

2,  Part  2,  114-115.    Bui.  Gembloux  (1890),  47,  1-17. 
"PR>^MOWSKI.     Die  Wurzelknollehen   der   Erbse.      (1)    Die   Atiologie  und 

Entwickelurigs-geschichte  der  Knollchen.     Landw.  Vers.  Stat.    (1890), 

37,  161-238. 
PRA£MOWSKI.     Die  Wurzelknollehen  der  Erbse.     Die  Biologische  Bedeutung 

der  Wurzelknollehen.    Landw.  Vers.  Stat.  (1890),  38,  5-62. 
PRILLIEUX.     Anciennes  observations  sur  les  tubercles  des  racines  des  L6gum- 

ineuses.    Compt.  Eend.  Acad.  Sci.  (Paris)    (1890),  111,  926-927.     Bot. 

Centbl.  (1891),  No.  8,  248. 
SCHLOESING.     Eemarques  an   sujet   des  observations   de  M.   Berthelot,   etc. 

Compt.  Eend.  Acad.  Sci.  (Paris)  (1890),  110,  612-613. 
SCHLOESING,  TH.  FILS,  ET  LAURENT.     Sur  la  fixation  de  1  'azote  gaseux  par 

les  Legumineuses.    Compt.  Eend.  Acad.  Sci.  (Paris)  (1890),  111,  750-754. 
WILFARTH.     Die  Stickstoffaufnahme  der  Pfianzen.    Versamml.  Deut.  Naturf. 

u.  Aerzte  zu  Bremen  (1890),  II,  63,  Sekt.  f.  Agrikulturchemie,  549-551. 
1891  ARCANGELI.     Sopra  i  tubercoli  radicali  delle  Leguminose.     Atti  B.  Accad. 

Lincei,  Eend.  Cl.  Sci.  Fis.,  Mat.  e  Nat.  (1891),  7,  223-227. 
ATWATER  AND  WOODS.     The  acquisition  of  atmospheric  nitrogen  by  plants. 

Amer.  Chem.  Jour.  (1891),  12,  526-547,  and  13,  42-51. 
BEYERINCK.     Over  Ophooping  van  Atmospherische  Stickstof  in  Culturen  van 

Bacillus  Eadicicola.     Vers.  en  Meded.  K.  Akad.  Wetensch.  (Amsterdam) 

(1891),  Afd.  Natmirk.  Ill,  8,  460-475. 


1917]   POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES       16? 

COHN.     Zur  Geschichte  der  Leguminosenknollchen.     Centbl.  f.  Bakt.  1  Abt. 

(1891),  10,  190. 
FRANK,  B.     Ueber  die  auf  Verdammg  von  Pilzen  abzielende  Symbiose  der 

mit  endotrophen  Mykorrhizen  begabten  Pflanzen,  sowie  der  Leguminosen 

und  Erlen.    Ber.  Deut.  Bot.  Gesell.  (1891),  9,  244-253. 
FRANK,  B.     Inwieweit  1st  der  freie  Luftstickstoff  fiir  die  Ernahrung  der 

Pflanzen  verwertbar?    Deut.  Landw.  Presse.  (1891),  18,  779-780. 
FRANK,  B.,  UND  OTTO.    Ueber  einige  neuere  Versuche  betreffs  der  Stickstoff- 

Assimilation  in  der  Pflanze.     Deut.  Landw.  Presse.  (1891),  18,  403-404. 
KIONKA.     Die  Wurzelknollchen  der  Leguminosen.    Biol.  Centbl.  (1891),  11, 

282-291. 
LAURENT.     Kecherches  sur  les  nodosites  radicales  des  Legumineuses.     Ann. 

Inst.  Pasteur  (1891),  5,  105-139. 
MORCK.    Ueber  die  Formen  der  Bakteroiden  bei  den  einzeln  Species   der 

Leguminosen.     Inaug.  Diss.  (Leipzig)    (1891),  Akademische  Buehhand- 

lung  (W.  Faber),  1-44. 
NOBBE.     Stickstoffernahrung  der  Leguminosen.     Chem.  Centbl.    (1891),  62, 

Part  1,  706. 

NOBBE,  SCHMIDT,  HILTNER,  UND  HOTTER.    Versuche  iiber  Stickstoff- Assimila- 
tion der  Leguminosen.    Landw.  Vers.  Stat.  (1891),  39,  327-359. 
OTTO.     Die  Assimilation  des  freien  atmospharischen  Stickstoffes  durch  die 

Pflanze.     Bot.    Centbl.    (1891),   46,    387-391,   and   47,    62-67,    123-129, 

175-180. 
SCHLOESING,  TH.  FILS,  ET  LAURENT.    Sur  la  fixation  de  1  'azote  par  les  plantes. 

Compt.  Rend.  Acad.  Sci.  (Paris)   (1891),  113,  776-778,  1059-1060. 
WILFARTH.    Stickstoff  auf  nahme  der  Pflanzen.    Landw.  Vers.  Stat.   (1891), 

38,  322-323.     Also  in  Chem.  Centbl.  (1891),  62,  Part.  I,  677. 
WOODS.     The  acquisition  of  nitrogen  by  growing  plants.     Conn.  Agr.  Exp. 

Sta.  Ann.  Ept.  (Storrs)   (1891),  4,  17-41. 
1892  ALPE  E  MENOZZI.     Studi    e    rieerche    sulla    questione    delle    assimilazione 

dell'azoto  per  parte  delle  piante.     Bui.  Not.  Agraris  Ministere  Agric. 

(1892),  No.  14.    Bot.  Centbl.  (1892),  51,  337. 
ATWATER  AND  WOODS.     The  fixation  of  free  nitrogen  by  plants.    Conn.  Agr. 

Exp.  Sta.  Ann.  Ept.  (Storrs)   (1892),  5,  17-22. 
DROBNIG.     Beitrage  zur  Kenntniss  der  Wurzelknollen.  Inaug.  Diss.  (Rostock) 

(1892).    Abst.  in  Bot.  Centbl.  2  Abt.  (1893),  56,  89-92.    Cited  in  Exp. 

Sta.  Eec.,  5,  434. 
FRANK,  B.      Die  Assimilation  freien  Stickstoffs  bei  den  Pflanzen  in  Ihrer 

Abhangigkeit    von     Species,     von     Ernahrungsverhaltnissen     und    von 

Bodenarten.     Landw.  Jahrb.  (1892),  21,  1-45. 
FRANK,  B.     Ueber  die  auf  den  Gasautausch  beziiglichen  Einrichtungen  und 

Thatigkeiten  der  Wurzelknollchen  der  Leguminosen.     Ber.  Deut.  Bot. 

Gesell.  (1892),  10,  271-281.      . 
FRANK,  B.     Ueber  den  Dimorphisumus  der  Wurzelknollchen  der  Erbse.    Ber. 

Deut.  Bot.  Gesell.  (1892),  10,  170-178,  390-395. 
GAUTIER  ET  DROUIN.     Remafques  sur  le  mecanisms  de  la  fixation  de  1 'azote 

par  le  sol  et  les  ve'getaux  a  propos  d'une  reponse  de  MM.  Schloesing, 

Fils,  et  Laurent.    Compt.  Rend.  Acad.  Sci.  (Paris)   (1892),  114,  19. 
IMMENDORFF.     Beitrage  zur  Losung  der  ' '  Stickstoff-Frage. ' '    Landw.  Jahrb. 

(1892),  21,  281-339. 
KOSSOWITSCH,  P.     Durch  welche  Organe  nehme  die  Leguminosen  den  freien 

Stickstoff  auf?     Bot.  Ztg.    (1892),  50,  697-702,   713-723,  729-738,  745- 

756,  771-774. 
LAWES  AND  GILBERT.     Sources  of  nitrogen  in  leguminous  plants.    Jour.  Roy. 

Agr.  Soc.  England   (1892),  2,  Ser.  3,  657-702. 


168  BULLETIN  No.  202  [July, 

MO'LLER.  Bemerkungen  zu  Frank's  Mittheilung  iiber  den  Dimorphismus  der 
Wurzelknollchen  der  Erbse.  Ber.  Deut.  Bot.  Gesell.  (1892),  10,  242- 
249,  568-570. 

•    NOBBE  UNO  HILTNER.     Ueber    die   Verbreitungsfahigkeit   der   leguminosen- 
bakterien  im  Boden.     Landw.  Vers.  Stat.   (1892),  41,  137-140. 

PETERMANN.  Contribution  a  la  question  de  1  'azote.  Mem.  Acad.  Roy.  Belg. 
(1892),  47,  Chem.  Centbl.  (1892),  Ser.  4,  4,  Part  2,  880-881. 

PROVE.  Untersuchungen  ueber  die  Stickstoffnahrung  der  Erbsen.  Ztschr. 
Landw.  Vers.  Bayern  (1892),  82,  85.  Centbl.  Agr.  Chem.  (1894),  23, 
404. 

SCHLOESING  ET  LAUBENT.  Recherches  sur  la  fixation  de  1 'azote  libre  par  les 
plantes.  Ann.  Inst.  Pasteur  (1892),  6,  65-115,  824-940. 

SCHLOESING  ET  LAURENT.  Sur  la  fixation  de  1 'azote  libre  par  les  plantes. 
Compt.  Rend.  Acad.  Sci.  (Paris)  (1392),  115,  659-661,  732-738.  Com- 
ments by  Duclaux  and  by  Berthelot  follow  these  references. 

^SCHNEIDER.  Observations  on  some  American  Rhizobia.  Bui  Torrey  Bot. 
Club  (1892),  19,  203-218. 

1893  "ATKINSON.     Contributions  to  the  biology  of  the  organism  causing  leguminous 

tubercles.     Bot.  Gaz.   (1893),  18,  157-166,  226-237,  257-266. 
BERTHELOT.      Recherches    nouvelles    sur    les    microorganismes    fixatures    de 

1 'azote.    Compt.  Rend.  Acad.  Sci.  (Paris)    (1893),  116,  847. 
BOLLEY.     Notes  on  root  tubercles  of  indigenous  and  exotic  legumes  in  virgin 

soil  of  the  Northwest.     Agricultural  Science  (1893),  7,  58-66. 
CLOS.     Revision  des  tubercles  des  rautes  et  des  tubercoloides  des  Legumi- 

neuses.     Mem.  Acad.  Sci.  Inscrip.  Belles-Lettres  Toulouse   (1893),  9th 

ser.,  5,  381-405.    Just 's  Bot.  Jahresber.     (1893),  21,  Part  I,  548. 
CONN.     Free  nitrogen  assimilation  by  plants.    Bui.  Torrey  Bot.  Club  (1893), 

20,  148-157. 
FRANK,    B.     Noch    ein   wort    zur   Stickstoff-Frage.      Deut.    Landw.    Presse 

(1893),  20,  183.    Jahresber.  Agr.  Chem.  (1893),  16,  98. 
FRANK,  B.     Die  Assimilation  des  freien  Stickstoffs  durch  die  Pflanzenwelt. 

Bot.  Ztg.,  1  Abt.  (1893),  51,  139-156. 
GAIN.     Influence  de  1'humidite  sur  le  developpement  des  nodosites  des  L6- 

gumineuses.     Compt.  Rend.  Acad.  Sci.   (Paris)    (1893),  116,  1394-1400. 
LIEBSCHER.     Beitrag  zur  Stickstoff-Frage.     Jour.  Landw.    (1893),  41,   139. 

Also  Centbl.  Agr.  Chem.  (1894),  23,  676-680. 
NOBBE  UNO  HILTNER.     Wodurch  werden  die  knollchen-besitzenden  Legum- 

inosen   befahight,    den    freien   atmospharischen   Stickstoff   fur    sich    zu 

verwerten?     Landw.  Vers.   Stat.    (1893),  42,   459-478. 
NOBBE  UND  HILTNER.     Eine  aus  der  ungleichen  Wirkungskraft  der  Knoll- 

chenbakterien    auf    die    verschiedenen    Leguminosen    sich    ergehbende 

praktische   Schlufsfolerung.     Sachs   Landw.    Ztscher.    (1893),   41,   595. 

Ref.  in  Jahresber.  Agr.  Chem.  (1893),  16,  197. 
"SCHNEIDER.     A  new  factor  in  economic  agriculture.    111.  Agr.  Exp.  Sta.  Bui. 

29   (1893),  301-319. 

SCHNEIDER.  The  morphology  of  root  tubercles  of  Leguminosae.  Amer.  Nat. 
(1893),  27,  782-792. 

WAGNER.  1st  es  wahr  dass  der  weisze  Senf  den  freien  Stickstoff  der  at- 
mospharischen Luft  aufnimmt  und  nach  Art  der  Leguminosen  sticks- 
toff  bereichernd  wirkt?  Deut.  Landw.  Presse  (1893),  20,  901-902,  913, 
933-934,  941-942. 

1894  BEYERINCK.     Ueber  die  Natur  der  Faden  der  Papilionaceenknollchen.    Centbl. 

f.  Bakt.,  1  Abt.  (1894),  15,  728-732. 

DUCLAUX.  Sur  la  fixation  de  1 'azote  atmosphe'rique.  Ann.  Inst.  Pasteur 
(1894),  8,  728-736. 


1917]   POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES       169 

FRANK,  B.  Neue  Stimmen  ueber  die  Stickstoffrage.  Deut.  Landw.  Presse 
(1894),  21,  119.  Jahrcsber.  Agr.  Chem.  (1894),  17,  172. 

GONNERMANN.  Die  Bakterien  in  den  Wurzelknollchen  der  Leguminosen. 
Landw.  Jahrb.  (1894),  23,  648-671. 

LECOMTE.  Les  tubercles  radicaux  de  1'Arachide.  Compt.  Eend.  Acad.  Sci. 
(Paris)  (1894),  119,  202-304. 

MACDOUGAL.  Titles  of  literature  concerning  the  fixation  of  free  nitrogen. 
Minn.  Bot.  Studies,  Bui.  9  (1894),  Part  4,  186-221. 

PETERMANN.  Contribution  a  la  question  de  1 'azote.  Recherches  Chim.  et 
Phys.  Appliquees  a  1'Agr.  Bruxelles-Paris  (1894),  2,  204-274.  Also 
Centbl.  Agr.  Chem.  (1894),  23,  674. 

RUSSELL.     The  fixation  of  free  nitrogen  by  plants.     Bot.  Gaz.   (1894),  19, 

284-293. 
SALFELD.     Die  Vernichtung  der  Leguminosenpilze   dureh   Aetzkalk.     Deut. 

Landw.  Presse  (1894),  21,  960.    Jahresber.  Agr.  Chem.  (1894),  17,  140. 

SCHNEIDER.     Beitrag  zur  Kenntniss  der  Ehizobien.     Ber.  Deut.  Bot.  Gesell. 

(1894),  12,  11-17. 
WARD.     Recent  investigations  and  ideas  on  the  fixation  of  nitrogen  by  plants. 

Nature  (1894),  49,  511. 

1895  BASSLER.     Sandkultur  versuche  ueber  die  Stickstoffassimilation  der  gelbcn 

Lupine  im  Sterilisierten  und  geimpften  Boden  bei  dargebot  wechselender 

Mengen   von   Saltpetersauren    Salzen.      Jahresber   Agr.   Chem.    (1895), 

38,  131. 
BILLWILER.     Ueber  Stickstoffassimilation  einigen  Papilionaceen,  deren  Bedeu- 

tung  fur    die  Landwirtschaft  unter  specieller  Beriicksichtigung  schwei- 

zerischer  Verhaltnisse.     Bot.  Centbl.   (1895),  63,  152. 
GAIN.     Action  de  1'eau  du  sol  sur  la  vegetation.     ReV.  Gen.  Bot.   (1895), 

7,  15-26. 
KIRCHNER.     Die  Wurzelknollchen  der  Soja-bohne.     Cohn's  Beitr.  Biol.  Pflan- 

zen  (Breslau)   (1895),  7,  Heft  2,  213-223. 

NOBBE  UND  HILTNER.  Vermogen  auch  Nichtleguminosen  freien  Stickstoff 
aufzunehmen?  Landw.  Vers.  Stat.  (1895),  45,  155-159. 

NOBBE,  HILTNER,  UND  SCHMIDT.  Versuche  ueber  die  Biologic  der  Knoll- 
chenbakterien  der  Leguminosen,  insbesondere  ueber  die  Frage  der  Artein- 
heit  derselben.  Landw.  Vers.  Stat.  (1895),  45,  1-27. 

SALFELD.  Die  Wirkung  von  Lehm  aus  dem  Untergrunde  und  von  Seeschlick 
und  die  Knollchen-Bakterien  der  Leguminosen.  Deut.  Landw.  Prcsse 
(1895),  22,  425. 

STOKLASA.  Studien  ueber  die  Assimilation  elementaren  Stickstoffs  durch  die 
Pflanzen.  Landw.  Jahrb.  (1895),  24,  827-863. 

STUTZER.  Nenere  Arbeiten  ueber  die  Knollchenbakterien  der  Leguminosen 
und  die  Fixerung  des  freien  Stickstoffs  durch  die  Thatigkeit  von  Micro- 
organismen.  Centbl.  f.  Bakt,  2  Abt.  (1895),  1,  68-74. 

DEVRIEZE.     Kann  man  mittelst  Lehm  Leguminosenpilze  einimpfen?     Deut. 

Landw.  Presse.     (1895),  22,  241. 
DEVRIEZE.     Vernichtung  der  Leguminosenpilze  durch  Aetzkalk  oder  Impfung 

der  Leguminosenpilze  durch  Velzener  Lehmmcrgel.    Deut.  Landw.  Presse. 

(1895),  22,  895. 
WARD.     New  aspects  of  an  old  agricultural  question.  Science  Progress  (1895), 

3,  251-271. 

1896  AEBY.     Beitrag  zur  Frage  der  Sticksto*ffernahrung  der  Pflanzen,     Landw. 

Vers.  Stat,  (1896),  46,  409-439. 


170  BULLETIN  No.  202 

CLOS.    CaractSres  exterieures  et  modes  de  repartition  des  petites  tubercles  ou 

tubereuloides    des    Le"gumineuses.     Compt.    Bend.    Acad.    Sci.    (Paris) 

(1896),  123,  407-410. 
CZAPEK.     Zur   Lelire    von    den    Wurzelausscheidungen.     Jahrb.    Wiss.    Bot. 

(1896),  29,  321-390. 
NAUDIN.    Nouvelles  recherches  sur  les  tubercules  des  Legumineuses.    Compt. 

Kend.  Acad.  Sci.  (Paris)  (1896),  123,  666-671. 
NOBBE.     Ueber  einige  neuere  Beobachtungen  betreffend  die  Bodenimpfung 

mit  rein  cultivirten  Knbllchenbakterien  fiir  die  Leguminosen  Cultur.    Bot. 

Centbl.  (1896),  68,  171-173. 
NOBBE  UNO  HILTNER.    Ueber  die  Anassungsfahigkeit  der  Knollchenbakterien 

ungleichen  Ursprungs  an  verschiedene  Leguminosengattungen.     Landw. 

Vers.  Stat.  (1896),  47,  257-268. 
STUTZEE.    Neuere  Arbeiten  ueber  die  Knollchenbakterien  der  Leguminosen 

und  die  Fixierung  des  freien  Stickstoffs  durch  Organismen.    Centbl.  f. 

Bakt.  2  Abt.  (1896),  2,  650-653. 
"STUTZER,  BURRI,  UND  MAUL.    Untersuchungen  ueber  Anpassungsvermogen 

von  Bacillus  radicicola  an  einen  f  remden  Nahrboden.  Centbl.  Bakt.  2  Abt. 

(1896),  2,  665-669. 
THIEL.     Mitteilung   ueber   die   Frage    der   Leguminosenknb'llchen.      Jahrb. 

Deut.  Landw.  Gesell.   (1896),  11,  48-52.    Koch's  Jahresber.  (1896>,  7, 

200. 

1897  HILTNER.     Ueber    Entstehung   und   physiologische   Bedeutung    der   Wurzel- 

knollchen.    Forstl.  Naturw.  Ztschr.  (1897),  23-36. 

MAZE.  Fixation  de  1 'azote  libre  par  la  bacille  des  nodositds  des  Le'gum- 
ineuses.  Ann.  Inst.  Pasteur  (1887),  11,  44-54. 

NOBBE.  Einige  neuere  Beobachtungen,  betreffend  die  Bodenimpfung  mit 
rein  cultivirten  Wurzelknollchen-Bakterien  fur  die  Leguminosen  Cultur. 
Verhandl.  Ges.  Deut.  Naturf.  Aerzte,  68  Versammlung  Frankfort  (1897), 
Halfte  1,  146-151.  See  also  Bot.  Centbl.  Beihefte  (1897),  7,  296. 

PFEIFFER  UND  FRANKE.  Beitrag  zur  f  rage  der  Verwertung  elementaren  Stick- 
stoffs durch  den  Senf.  Landw.  Vers.  Stat.  (1897),  48,  455-467. 

ZINSSER.  Ueber  das  Verhalten  von  Bakterien,  insbesondere  von  Knb'llehen- 
bakterien,  in  lebenden  pflanzlichen  Geweben.  Jahrb.  Wiss.  Bot.  (1897), 
30,  423-452. 

1898  FREMLIN.     Organisms  in   the  nodules  on  the  roots  of  leguminous  plants. 

Jour.  Path,  and  Baet.  (1898),  5,  389-398. 
MAZE.    Les  microbes  des  nodosite"s  des  Legumineuses.     Ann.  Inst.  Pasteur 

(1898),  12,  No.  1,  1-25,  128-155. 
MOTTOREALE.    Di  alcuni  tubercolifere  dello  Hedysarum  coronarium  in  rela- 

zoine  al  Bacileus  radicicola  e  alia  Phytomyxa  leguminosarum.  Atti  R.  1st. 

Ineoraggiamen  to  Napoli  (1898),  ser.  4,  11,  1-7. 

NOBBE  UND  HILTNER.  Ueber  die  Dauer  der  Anpassungsfahigkeit  der  Knoll- 
chenbakterien an  bestimmte  Leguminosarten.  Landw.  Vers.  Stat.  (1898), 

49,  467-480.    Bot.  Centbl.,  Beihefte  (1898),  8,  64. 
SCHWAN.     Ueber  das  Vorkommen  von  Wurzelbakterien  in  abnorm  verdickten 

Wurzeln  von  Phaseolus  multiflorus.  Inaug.  Diss.  (Erlangen)   (1898).  See 

also  Just's  Bot.  Jahresber.  (1898),  26,  135. 

1899  CLOSE.    Les  tuberculoids  des  Le"gumineuses  d'apres  Charles  Naudin.     Bui. 

Soc.  Bot.  France  (1899),  6,  Ser.  3,  396-403. 
*°GREIG-SMITH.     The  nodule  organism  of  the  Leguminosae.    Proc.   Linn.  Soc. 

N.  S.  Wales  (1899),  24,  653-673.    See  also  Centbl.  f.  Bakt,  2  Abt.  (1900), 

6,  371-372. 
HILTNER.  Ueber  die  Assimilation  des  freien  atmospharischen  Stickstoffs  durch 

in  oberirdischen  Pflanzenteilen  lebende  Mycelien.     Centbl.  f.  Bakt.  2 

Abt.  (1899),  5,  831-837, 


1917]   POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES       171 

MATTIROLO.  Sulla  influenza  che  la  estirpazione  die  fiori  esercita  suit  tuber- 
coli  radical!  delle  piante  Leguminose.  Malpighia  (1899),  13,  382-421. 

MMAZE.  Les  microbes  des  nodosite"s  des  Le*gumineuses.  Ann.  Inst.  Pasteur 
(1899),  13,  145. 

NOBBE  UNO  HILTNER.  Ueber  die  Wirkung  der  Leguminosenknollchen  in  der 
Wasserkultur.  Landw.  Vers.  Stat.  (1899),  52,  455-465. 

PARATORE.  Ricerche  istologiche  sui  tubercoli  radicali  delle  Leguminose.  Mal- 
pighia (1899),  13,  211-230. 

EICHTER.  Zur  Frage  der  Stickstoffernahrung  der  Pflanzen.  'Landw.  Vers. 
Stat.  (1899),  51,  221-241. 

STOKLASA  UNO  SEMPOLOWISKI.  Versuche  mit  Nitragin  und  Alinit.  Deut. 
Landw.  Presse  (1899),  26,  13-14. 

1900  22DAWSON.     "Nitragen"  and  nodules   of  leguminous  plants.     Phil.   Trans. 

Roy.  Soc.  London  (1900),  Ser.  B,  192,  1-28. 
^DAWSON.     Further  observations  on  the  nature  and  functions  of  the  nodules 

of  leguminous  plants.     Phil.  Trans.  Roy.  Soc.  London,   (1900),  Ser.  B. 

193,  51-67. 
DEHERAIN  ET  DEMOUSSY.     Sur  la  cultures  des  Lupins  bleus.    Compt.  Rend. 

Acad.  Sci.  (Paris)   (1900),  130,  20-24. 

DEHERAIN  ET  DEMOUSSY.    Culture  des  Lupins.  Ann.  Agron.  (1900),  26,  57-77. 
DEHERAIN  ET  DEMOUSSY.     Recherches  sur  la  Ve'ge'tation  de  Lupins.  Deuxieme 

partie:     Lupins  bleus   (Lupinus  angustifolius).     Ann.  Agron.   (1900), 

26,  169-196. 
HARTLEB.     Die   Morphologie   und  systematische    Stellung  der  sogenannten 

Knollchenbakterien.    Chem.  Ztg.  (1900),  24,  887-888. 
HILTNER.     Ueber  die  Ursachen  welche  die  Grosse,  Zahl,  Stellung,  und  Wirkung 

der  Wurzelknollchen  der  Leguminosen  bedingen.    Arb.  K.  Gsndhtsamt., 

Biol.  Abt.    (1900),  1,  Heft  2,  177.     Abst.  in  Just's  Bot.  Jahresber. 

(1900),  28,  Part  1,  47. 
HILTNER.     Ueber    die    Bakteroiden    der    Leguminosenknollchen    und    ihre 

wirkiirliche  Erzeugungausserhalb  der  Wirtspflanzen.     Centb.  f.  Bakt.  2 

Abt.  (1900),  6,273-281. 

LUTOSLAWSKI.  Beitrag  zur  Lehre  von  der  Stickstoffernahrung  der  Legum- 
inosen. Ber.  Landw.  Inst.  Univ.  Halle.  (1900),  Hefte  14,  36.  See  also 

Jahresber.  Agr.  Chem.  (1900),  43,  125-126. 
"NOBBE  UND  HILTNER.     Kunstliche  Ueberf  iihrung  der  Knollchenbakterien  von 

Erbsen  in  solche  von  Bohnerr.     (Phaseolus).     Centbl.  f.  Bakt.  2  Abt. 

(1900),  6,  449-457. 
STUTZER,     Beitrage  zur  Morphologie  der  als  "Bacterium   radicicola"  be- 

schriebenen  Organis'men.     Mitt.  Landw.  Inst.  K.  Univ.  Breslau  (1900), 

Heft.  Ill,  57-71.    Just's  Bot.  Jahresber.  (1900),  28,  38. 

1901  BURRAGE.     Description  of  certain  bacteria  obtained  from  nodules  of  various 

leguminous  plants.  (A  preliminary  study  on  the  constancy  of  the  dis- 
tribution of  bacterial  species  in  definite  species  of  leguminous  plants.) 
Proc.  Ind.  Acad.  Sci.  (1901),  157-161. 

BURRI.  Die  Stickstoffernahrung  der  Leguminosen  und  die  Knollchenbak- 
terien. Schweiger  Landw.  Centbl.  Frauenfeld  (1901),  21,  97-112,  139- 
150. 

CONN.     Agricultural  bacteriology,  2d.  ed.  (1909),  93-110.    (Philadelphia) 

DAWSON.  On  the  economic  importance  of  "Nitragin. "  Ann.  Bot.  (1901), 
15,  511-519. 

GREIG-SMITH.  The  nature  of  the  bacteroids  of  the  leguminous  nodule  and 
the  culture  of  Rhizobium  leguminosarum.  Proc.  Linn.  Soc.  N.  S.  Wales 
(1901),  36,  152-155. 

LAURENT.  Observations  sur  le  deVeloppement  des  nodosite"s  radieales  chez 
les  Le"gumineuses.  Compt.  Bend.  Acad.  Sci.  (Paris)  (1901),  133,  1241- 
1243. 


172  BULLETIN   No.  202  [July, 

MARCHAL.     Influence  cles  sels  min^raux  nitritifs  sur  la  production  des  nodo- 

sites  chez  le  pois.    Compt.  Rend.  Acad.  Sci.  (Paris)    (1901),  133,  1032- 

1033. 
NOBBE   UNO   HILTNER.     Ueber   den   Einfluss   verschiedener    Impstoffmengen 

auf  die  Knollchenbildung  und  den  Ertrag  von  Leguminosen.     Landw. 

Vers.  Stat.  (1901),  55,  141-148. 
PARATORE.     Eicerche  sulla  struttura  e  le  alterazioni  del  nucleo  nei  tubercoli 

radical!  delle  Leguminose.     Malpighia   (1901),  15,  178-187.     Centbl.  f. 

Bakt.  2  Abt.  (1902),  8,  715. 
PARATORE.     Sul  polimorfismo  del  Bacillus  Eadicicola  Bey.    Malpighia  (1902), 

15,  175.     Centbl.  f.  Bakt.  2  Abt.  (1902),  8,  715. 
PASSERINI.     Sui  tubercoli  radical!  della  Medicago  sativa  L.     Bui.  Soc.  Bot. 

Ital.  (1901),  N  8,  365-370. 
STUTZER.     Die  Bildimg  von  Bakteroiden  in  Kiinstlichen  Nahrboden.    Centbl. 

f.  Bakt.  2  Abt.  (1901),  7,  897-912. 

1902  BREFELD.     Versuche  ueber  die  Stickstoffaufnahme  bei  den  Pflanzen.    Centbl. 

f.  Bakt.  2  Abt.  (1902),  8,  24-25. 
BUHLERT.     Ein  weiterer  Beitrag  zur  Frage  der  Artenheit  der  Knollchenbak- 

terien  der  Leguminosen.    Centbl.  f.  Bakt.  2  Abt.  (1902),  892-895. 
BUHLERT.     Untersuchungen  ueber  die  Artenheit  der  Knollchenbakterien  der 

Leguminosen  und  Ueber  die  landwirtschaftliche  Bedeutung  dieser  Frage. 

Fiihling's  Landw.  Ztg.     (1902),  51,  Heft.  11,  385-391,  and  Heft.  12, 

417-427.     See  also  Centbl.  f.  Bakt.   (1902)  2  Abt.,  9,  148-153,  226-240, 

273-285. 
HILTNER.     Ueber  die  Impfung  der  Leguminosen  mit   Reinkulturen.     Dent. 

Landw.  Presse  (1902),  29,  No.  15,  119-120. 

HOPKINS     Alfalfa  on  Illinois  soil.    111.  Agr.  Exp.  Sta.  Bui.  76  (1902). 
MOORE.     Bacteria  and  the  nitrogen  problem.     U.  S.  Dept.  Agr.  Yearbook 

(1902),  333-342. 
NEUMANN.     Die  Bakterien  der  Wurzelknollchen  der  Leguminosen.     Landw. 

Vers.  Stat.  (1902),  56,  187-206. 
NEUMANN.     Untersuchungen    ueber    das    Vorkommen    von    Sti.ckstoff-assim- 

ilierenden  Bakterien  im  Ackerboden.     Landw.  Vers.   Stat.    (1902),  56, 

203-206. 
NOBBE  UND  RICHTER.     Ueber  den  Eiufluss  des  Nitrastickstoffs  und  der  Huirnis- 

substanzen  auf  den  Impfungserfolg  bei  Leguminosen.     Landw.   Vers. 

Stat.  (1902),  56,  441-448. 
^PEIRCE.     The  root  tubercles  of  Bur  clover   (Medicago  denticulata  WilM), 

and  of  some  other  leguminous  plants.    Proc.  Cal.  Acad.  Sci.  (1902),  3d. 

Ser.,  2,  (Botany),  295-328. 
REMY.     Ueber  die   Steigerung  des  Stickstoffsammlungsvermb'gens  der  Hiil- 

senfriichte  durch  bakterielle  Hilfsmittel.     Deut.  Landw.  Presse   (1902), 

29,  No.  5,  31-32,  and  No.  7,  46-48. 
SCHNEIDER.     Contribution  to  the  biology  of  Rhizobia :  I,  Rhizobium  mutabile 

in  artificial  culture  media.    Bot.  Gaz.  (1902),  34,  109-113. 
WOHLTMANN.     Die  Knollchen-Bakterien  in  ihrer  Abhanghigkeit  von  Boden 

und  Diingung.    Jour.  Landw.  (1902),  50,  377-395. 

1903  GOLDING.     Experiments  on  peas  in  water  cultures.     Centbl.  f.  Bakt.  2  Abt. 

(1903),  11,  1-7. 
HILTNER  UND   STORMER.     Neue  Untersuchimgen   ueber   die  Wurzelknollchen 

der  Leguminosen  und  deren  Erreger.     Arb.  K.  Gsndhtsamt.,  Biol.  Abt. 

(1903),  3,  Heft.  3,  151-307. 

I'ETRI.     Di  tin  nuovo  bacillo  capsulato  e  del  significato  biologico  delle  capsule. 
Nuove  Giornale  Botanico   (1903),   (2)   X,  272.     Centbl.  f.  Bakt.  2  Abt. 

(1903),  11,  347. 


1917]   POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES        173 

REMY.     Stickstoffbindung  (lurch  Leguminoscn.     Verb.  Ges.  Naturf.  Leipzig 

(1902),  74,  I,  (1903),  200-221.    Centbl.  f.  Bakt.  2  Abt.  (1904),  12,  498. 
SCHNEIDER.     Outline  of  the  history  of  leguminous  root  nodules  and  Bhizobia, 

with  titles  of  literature  concerning  the  fixation  of  free  nitrogen  by  plants. 

Minn.  Bot.  Studies  (1903),  ser.  3.,  2,  133-139. 
SCHNEIDER.     Contribution  to  the  biology  of  Ehizobia:     II,  The  motility  of 

Rhizobium  mutabile.    Bot.  Gaz.  (1903),  35,  56-58. 
SCHNEIDER.     Contribution  to  the  biology  of  Rhizobia:     III,  Notes  on  the 

winter  and  early  spring  conditions  of  leguminous  root  tubercles.     Bot. 

Gaz.  (1903),  36,  64-07. 

1904  FLAMAND.     De  1 'influence  de  la  nutrition  sur  le  developpement  des  nodositfis 

des  Legumineuses.     Ing6n.     Agr.  Gembloux  (1904),  4th  Annee,  755-765. 

Ref.  in  Centbl.  f.  Agr.  Chem.  (1905),  34,  738. 
HILTNER.     Bericht  ueber  die  Ergebnisse  der  im  Jahre  1903  in  Bayern  auf 

gefiihrten  Impfversuche  mit  Reineulturen  von  Leguminosen  Knollchen- 

bakterien    (Nitragin).      Naturw.   Ztschr.   Land.   u.   Forstw.    (1904),   2, 

Heft  3,  127-159. 
HILTNER.     Die  Bindung  von  freien  Stickstoff  durch  das  Zusammenwirken 

von  Schizomyceten  und  von  Eumyceten  mit  hoheren  Pflanzen.     Handb. 

der  Technischen  Mykologie,  Lafar,  2  Auflage,  (Jena),  (1904),  3,  24-70. 
HILTNER.     Recent  experiments  and  Dew  problems  in  soil  bacteriology,  with 

especial  reference  to  green  manuring  and  fallows.    Cited  in  Exp.  Sta. 

Rec.  (1904),  16,  546.    Arb.  Deut.  Landw.  Gesell.  (1904),  98,  59-78. 
"HOPKINS.     Nitrogen  bacteria  and  legumes.     111.  Agr.  Exp.   Sta.  Bui.   94 

(1904). 
27LEMMERMANN.     Untersuchungen  ueber  einige  Ernahrungsunterschiede  der 

Leguminosen  und  Gramineen  und  ihre  wahrscheinliche  Ursache.  Verhandl. 

Gesell.  Deut.  Naturf.  u.  Aerzte  (1904),  76,  II,  1,  145.  Landw.  Vers.  Stat. 

(1904),  67,  207-251. 
NOBBE  UND  RICHTER.     Ueber  den  Einfluss  des  in  Kulturboden  vorhanderen 

assimilierbaren    Stickstoffs    auf    die    Aktion    der    Knollchenbakterien. 

Landw.  Vers.  Stat.  (1904),  59,  167-174. 
REMY.     Neue  Untersuchungen  ueber  die  Knollchen  bakterien  der  Hu'lsen- 

fruchte.    Landbote  Prenzlau  (1904),  25,  366-386. 
SUCHTING.     Kritische  Studien  ueber  die  Knollchenbakterien.    Centbl.  f .  Bakt. 

2  Abt.  (1904),  11,  377-388,  417-441,  496-520. 

1905  CLARK.     Suggestions  concerning  legume  inoculation.     Mich.  Agr.  Exp.  Sta. 

Bui.  231  (1905). 
FITTING.     Ueber  die  Wurzelknollchenbakterien  als  Vermittler  der  Stickstoff- 

ernahrung  bei  Leguminosen.    Stuttgart,  Jahreshefte  Ver.  Natk.  (1905), 

61,  78-80. 
GOLDING.     The  importance  of  the  removal  of  the  products  of  growth  in  the 

assimilation  of  nitrogen  by  the  organisms  of  the  root  nodule  of  legumin- 
ous plants.    Jour.  Agr.  Sci.  (1905),  1,  59-64. 
GRUNER.     Utilizzazione  dell  'azote  atmosferico.     Ann.  Sci.  Indus.    (Milano) 

(1905),  42,  180-187. 
HARDING  AND  PRUCHA.     The  quality  of  commercial  cultures  for  legumes.    New 

York  (Geneva)  Agr.  Exp.  Sta.  Bui.  270  (1905). 
HEINZE.     Einige  Berichtigungen  und  weitere  Mitteilungen  zu  der  Abhand- 

lung,  ' '  Ueber  die  Bildung  und  Wiederverarbeitung  von  Glycogen  durch 

niedere  Pflanzliche  Organismen. "     Centbl.  f.  Bakt.  2  Abt.   (1905),  14, 

75-87,  168-183'. 
LEWIS  AND  NICHOLSON.     Soil  inoculation.    Tubercle  forming  bacteria  of  the 

legumes.    Okla.  Agr.  Exp.  Sta.  Bui.  68  (1905),  1-30. 
LO'HNIS.     Beitrage  zur  Kenntnis  der  Stickstoffbakterien.     Centbl.  f.  Bakt.  2 

Abt.  (1905),  14,  582-604,  713-723. 


174  BULLETIN  No.  202  [July, 

MMOOKE.     Soil  inoculation  for  legumes;  with  reports  upon  the  successful  use 

of  artificial  cultures  by  practical  farmers.    U.  S.  Dept.  Agr.  Bur.  Plant 

Indus.  Bui.  71  (1905). 
PEGLION.     Un 'esperienza  cogli  azotofogi  di  Moore.     Le  Stazioni  Speriment- 

alli  Agraria  Italiane,  Modena  (1905),  38,  769-784.     Abst.  in  Centbl.  f. 

Bakt.  2  Abt.  (1907),  18,  524. 
PEROTTI.     Influenza  di  alcune  azione  oligodinamiche  sullo  sviluppo  e  sull  'atti- 

vita  del  B.  radicicola  (Beyerinck).    Annali  di  Botanica  (1905),  3,  513- 

526. 
SCHNEIDER.     Contribution  to  the  biology  of  Bhizobia.  IV,  Two  coast  Khizobia 

of  Vancouver  Island,  B.  C.    Bot.  Gaz.  (1905),  40,  135-139. 
SMITH  AND  EOBINSON.     Observations  on  the  influence  of  nodules  on  the  roots 

upon  the  composition  of  soybeans  and  cowpeas.  Mich.  Agr.  Exp.  Sta.  Bui. 

224  (1905). 
VOORHEES  AND  LiPMAN.     Atmospheric  nitrogen  in  the  soil,  -experiments  on 

the  accumulation  and  utilization.    N.  J.  Agr.  Exp.  Sta.  Bui.  180  (1905). 
VUILLEMIN.    Hyphoides  et  bacteroides.     Compt.  Eend.  Acad.  Sci.   (Paris) 

(1905),  140,  52. 
WOODS     Inoculation  of  soil  with  nitrogen-fixing  bacteria.     U.  S.  Dept.  Agr. 

Bur.  Plant  Indus.  Bui.  72  (1905),  24-30. 

1906  BALL.     Nitro-culture.    Texas  Agr.  Exp.  Sta.  Bui.  83  (1906). 
BOTTOMLEY.     The  cross  inoculation  of  Leguminosae  and  other  root-nodule 

bearing  plants.    Ept.  Brit.  Assoc.  Adv.  Sci.  (1906),  76,  752-753. 
GREIG-SMITH.     The  formation  of  slime  or  gum  by  Ehizobium  leguminosarum. 

Proe.  Linn.  Soc.  N.  S.  Wales  (1906),  31,  264-294. 
GREIG-SMITH.     Structure  of  Ehizobium  leguminosarum.    Proe.  Linn.  Soe.  N. 

S.  Wales  (1906),  31,  295-302. 
GREIG-SMITH.     The  fixation  of  nitrogen  by  Ehizobium  leguminosarum.    Proe. 

Linn.  Soc.  N.  S.  Wales  (1906),  31,  608-615. 
HEINZE.     Ueber  die  Stickstoffassimilation  durch  niedere  Organismen.  Landw. 

Jahrb.  (1906),  35,  890-891. 
JAMIESON.    Utilization  de  1 'azote  de  1'air  par  les  plantes.    Ann.  Sci.  Agron. 

(1906),  3,  61-132.    Centbl.  Agr.  Chem.  (1907),  36,  387. 
KELLEKMAN.     Conditions  affecting  legume  inoculation.    U.  S.  Dept.  Agr.  Bur. 

Plant  Indus.  Bui.  100  (1906),  Part  8. 
KELLERMAN  AND  BECKWITH.     The  effect  of  drying  upon  legume  bacteria. 

Science  (1906),  N.  S.  23,  471-472. 
KELLERMAN  AND  BECKWITH.    Die  Bakterien  der  Wurzelknb'tchen  der  Legum- 

inosen.    Centbl.  f.  Bakt.  2  Abt.  (1906),  16,  540. 

LAFAR.     Handbuch  der  Technischen  Mykologie.  (1904-1906),  3,  24-71.  (Jena) 
^MAASSEN  UNO  MULLER.    Ueber  die  Bakterien  in  den  Knollchen  der  ver- 

schiedenen  Leguminosenarten.     Mitt.  a.  d.  Biol.  Anstalt.  f.  Land,  und 

Forstw.,  (1906),  2,  24.    Cited  by  Simon,  Centbl.  f.  Bakt.  2  Abt.  (1914), 

41,  471. 
SACKETT.     The  association  of  Ps.  radicicola  with  Bacillus  ramosus.     Ept. 

Mich.  Acad.  Sci.  (1906),  8,  147-150. 
STEFAN.     Studien  zur  Frage  Leguminosenknollchen.    Centbl.  f.  Bakt.  2  Abt. 

(1906),  16,  131-149. 
VOGEL.     Die  Assimilation  des  freien  elementaren  Stickstoffes  durch  Mikro- 

organismen.    Centbl.  f.  Bakt.  2  Abt.  (1905),  15,  174-188,  215-227. 

1907  BOTTOMLEY.     The  structure  of  root  tubercles  in  leguminous  and  other  plants. 

Ept.  Brit.  Assoc.  Adv.  Sci.  (1907),  77,  693. 

CHESTER.    .The  effect  of  desiccation  on  root  tubercle  bacteria.    Del.  Agr.  Exp. 
Sta.  Bui.  78  (1907). 


1917]   POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES       175 

10DE  Eossi.     Ueber  die  Microorganismen  welche  die  Wurzelknollchen  der  Le- 

guminosen  erzeugen.     Centbl.  f.  Bakt.  2  Abt.  (1907),  18,  289-314,  481- 

488. 
GREIG-SMITH.     The  fixation  of  nitrogen  by  the  nodule  former.     Jour.  Soc. 

Chem.  Indus.  (1907),  26,  304-306. 
"GROSBUSCH.  Ehizobium  radicicola  Usw.  Inaug.  Diss.  (Bonn)  (1907).  See  also 

Lohnis  Handbuch  der  landwirtsehaflichen  Bakterologie  (1910),  671. 
32HARRisoN  AND  BARLOW.     The  nodule  organism  of  the  Leguminosae,  its  isola- 
tion, cultivation,  identification,  and  commercial  application.     Centbl.  f. 

Bakt.  2  Abt.  (1907),  19,  264-272,  426-441.    Trans.  Eoy.  Soc.  Can.  (1907), 

Ser.  2,  12,  157-237. 
KELLERMAN  AND  FAWCETT.     Movements  of  certain  bacteria  in  soils.    Science 

(1907),  N.  S.  25,  806. 
MASSEN  UNO  BEHM.    Ueber  die  Bakterien  in  den  Knollchen  der  verschied- 

enen  Leguminosenarten.    Mitt.  Biol.  Anst.  Land.  Forstw.  (1907),  4,  42. 

Just's  Bot.  Jahresber.  (1907),  35,  Part  1,  474. 
EODELLA.     I  batterii  radicale  delle  Leguminose.    Studio  critico  sperimentale 

d'alcume  problemi  di  bacteriologia  agraria  e  di  fisiopatologia  unmana. 

Padova,  Prosperini  (1907),  1-87.    Centbl.  f.  Bakt.  2  Abt.  (1907),  18,  455. 
SEVERINI.     Bicerche  bacteriologiche  sui  tubercoli  radicali  di  Hedysarum  coro- 

narium  L.    (Sulla).  Atti  E.  Accad.  Lincei,  Eend.  Cl.  Sci.  Fis.,  Mat.  e  Nat. 

(1907),  Fasc.  3,  2  Sera.,  16,  219-226.    Centbl.  f  Bakt.  2  Abt.  (1908),  21, 

164. 
SIMON.     Die    Widerstandsfahigkeit    der   Wurzelbakterien    der   Leguminosen 

und  ihre  Bedeutung  fur  die  Bodenimpfung.    Jahresber.  Ver.  Augen  Bot. 

(1907),  5,  132-160.     Koch's  Jahresber.  (1907),  18,  436. 
VOORHEES  AND  LiPMAN.     A  review   of  investigations  in   soil  bacteriology. 

U.  S.  Dept.  Agr.  Office  Exp.  Sta.  Bui.  194  (1907),  87-107. 
WOODS.     The  present  status  of  the  nitrogen  problem.    U.  S.  Dept.  Agr.  Year- 
book (1906),  125-136. 

1908  BREFELD.     Die  Anwendung  der  Kulturmenthoden  fiir  die  verschiedenen  Pilz- 

formen.    Untersuch.  Gesamtgebiet  der  Mykologie  (1908),  14,  98. 
FRED.     Eesults  obtained  from  inoculating  soybeans  with  artificial  cultures. 

Vir.  Agr.  Exp.  Sta.  Ann.  Ept.  (1908),  130-131. 
FRED.     Assimilation  of  nitrogen  by  different  strains  of  Bacillus  radicicola 

in  the  absence  of  the  host  plant.    Vir.  Agr.  Sta.  Ann.  Ept.  (1908),  132. 
GERLACH  UND  VOGEL.     Beobachtungen  ueber  die  Wirkung  der  Hiltnerischen 

Eeinkulturen  fiir  Leguminosen.     Centbl.   f.  Bakt.  2  Abt.    (1908),  20, 

61-71. 
HARDING.     Bacteriological  investigations.     New  York    (Geneva)   Agr.  Exp. 

Sta.  Ann.  Ept.  (1908),  110-118. 
LAURENT.     Sur  le  microbe  des  nodosit^s  des  Le'gumineuses.    Becueil  de  Inst. 

Bot.  Univ.  Bruxelles  Public  Errera  (1908),  4,  83-85.    Centbl.  f.  Bakt.  2 

Abt.  (1910),  25,  506. 
LAURENT.     Eecherches  sur  les  nodosite's  radicales  des  Le'gumineuses.    Eecueil 

de  Inst.  Bot.  Univ.  Bruxelles  Publi6  Errera  (1908),  4,  87-126.    Centbl.  f. 

Bakt.  2  Abt.  (1910),  25,  506. 
NOBBE,  EICHTER,  UND   SIMON.     Untersuchungen    ueber    die    wechselseitige 

Impfung    verschiedener    Leguminosengattungen    mit    Eeinkulture    von 

Knollchenbakterien.    Landw.  Vers.  Stat.  (1908),  68,  229,  241. 
STEVENS  AND  TEMPLE.     The  efficiency  of  pure  culture  inoculation  for  legumes. 

N.  C.  Agr.  Exp.  Sta.  13th  Ann.  Ept.  (1908),  48-57. 

1909  BALL.     A  contribution  to  the  life  history  of  Bacillus  (Ps.)  radicicola  beij. 

Centbl.  f.  Bakt.  2  Abt.  (1909),  23,  47-59. 

BOTTOMLEY.     Some  effects  of  nitrogen  fixing  bacteria  on  the  growth  of  non- 
leguminous  plants.    Proc.  Eoy.  Soc.  London  (1909)  81,  B,  287. 


176  BULLETIN   No.  202  [July, 

BOTTOMLEY.     Nitrogen-fixing  bacteria  and  non-leguminous   plants.     Nature 

(1909),  82,  218. 
BUCHANAN.     The  gum  produced  by  Bacillus  radicicola.     Centbl.  f.  Bakt.  2 

Abt,  (1909),  22,  371-395. 
BUCHANAN.     The  bacteroids  of  Bacillus  radicicola.    Centbl.  f.  Bakt.  2  Abt. 

(1909),  23,  59-91. 
MDE  Bossi.     Studi  sul  microrganismo  produttore  dei  tubercoli  delle  Legum 

inose.    I — Isolamento,  diagnose  batteriologica,  utilizzazione  delle  culture 

nella  practica  agricola.    Annali  di  Botanica  (1909),  7,  Fasc.  4,  618-652. 

Centbl.  f.  Bakt  2.  Abt.  (1910),  26,  263-270. 
DE  Eossi.       Studi  sul  microrganismo  produttore  dei  tubercoli  delle  Legum- 

inose.      II — Sulle   fissazione    dell 'azote    elementare    nelle    culture   pure. 

Annali  di  Botanica  (1909),  7,  Fasc.  4,  653-669.    Centbl.  f.  Bakt.  2  Abt. 

(1910),  26,  270-272. 
EDWARDS  AND  BARLOW.     Legume  bacteria.    Further  studies  of  the  nitrogen 

accumulation  in  the  Leguminosae.     Ontario  Agr.  Col.  and  Exp.  Farm 

Bui.  164  (1909),  1-32,  45. 

HALL.     The  fixation  of  nitrogen  by  soil  bacteria.    Nature  (1909),  81,  98. 
HALL.     Nitrogen  fixing  bacteria  and  non-leguminous  plants.    Nature  (1909), 

82,  218-219. 
HENRI.     Sur  une  theorie  nouvelle  de  la  captation  de  1 'azote  atmospherique. 

Bui.  Soc.  Sc.  Nancy  (1909),  Ser.  3,  1-29.    Centbl.  f.  Bakt.  2  Abt.  (1910), 

27,  634. 
MAIRE  ET  TISON.     Phytomyxa  leguminosarum  (Frank)  Schrb'ter,  in  la  cyto- 

logie  des  plasmodiophoracees  et  la  classe  des  Phytomyxinae.    Ann.  Mycol. 

(1909),  7,  241. 
1910  BOTTOMLEY.     Nitrogen-fixing  bacteria  and  non-leguminous  plants.     Nature 

(1910),  83,  96. 
BOTTOMLEY.     The  fixation  of  nitrogen  by  free-living  soil  bacteria.    Bpt.  Brit. 

Assoe.  Adv.  Sci.  (1910),  581-582. 
FRED.     The  infection  of  root  hairs  by  means  of  Bacillus  radicicola.    Vir  Agr. 

Exp.  Sta.  Ann.  Ept.  (1909-10),  123. 
FRED.     The  fixation  of  nitrogen  by  means  of  Bacillus  radicicola  Avithout  the 

presence  of  a  legume.    Vir.  Agr.  Exp.  Sta.  Ann.  Ept.  (1909-10),  138. 
GAGE.     Biological  and  chemical  studies  on  nitroso  bacteria.    Centbl.  f.  Bakt. 

2  Abt.  (1910),  27,  7-48. 

GOLDING.     Notes  on  the  nature  of  nitrogen  fixation  in  the  root  nodules  of  le- 
guminous plants.     Ept.  Brit.  Assoc.  Adv.  Sci.  (1910),  80,  582. 
HILTNER.     Ueber  die  Impfung  der  Serradella  und  anderer  Kulturpflanzen  mit 

mehreren  Bakterienarten.    Illustr.  Landw.  Ztg.  (1910),  27,  634-635. 
"HOPKINS.     Soil  fertility  and  permanent  agriculture.     (1910),  208. 
KELLERMAN.     Nitrogen  gathering  plants.    TJ.  S.  Dept.  Agr.  Yearbook  (1910), 

213-218. 
KELLERMAN.     Methods  of  legume  inoculation.    U.  S.  Dept.  Agr.  Bur.  Plant 

Indus.  Cir.  63  (1910),  1-5. 
KELLERMAN.     Legume  inoculation  and  the  litmus  reaction  of  soils.     U.  S. 

Dept.  Agr.  Bur.  Plant  Indus.  Cir.  71  (1910). 
KELLERMAN.     Geisselfarbung  bei  Pseudomonas  radicicola.     Centbl.  f.  Bakt. 

2  Abt.  (1910),  27,  233.    Abst.  in  Science  (1910),  N.  S.  31,  554. 
LIPMAN.     Soil  inoculation  tests  of  commercial  cultures.    N.  J.  Agr.  Exp.  Sta. 

Bui.  227  (1910). 
LOHNIS.     Handbuch  der  landwirtschaftlicheu  Bakteriologie   (1910),  643-671. 

(Berlin) 
MAASSEN  UND  SCHO'NWALD.     Das  Verhalten  der  Bakterien  in  einer  Stick- 

oxydulatmosphare.     Mitt.  K.  Biol.  Anst.  Landw.  Forst.    (1910),  Heft  10, 

32.    Centbl.  f.  Bakt.  2  Abt.  (1910),  27,  636. 


1917]   POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES       177 

PEKLO.     Die  pflanzlichen  Aktinoinykosen.     Centbl.  f.  Bakt.  2  Abt.   (1910), 

27,  451-580. 

PERCIVAL.     Agricultural  bacteriology   (1910),  187-206.    (London). 
SANI.     Bicerche  chimicofisiologiche  sui  tubercoli  radical!  della  Vicia  faba. 

Eend.  Accad.  Lincei  (1910),  Ser.  5,  19,  207-211.    Centbl.  f.  Bakt.  2  Abt. 

(1911),  30,  75. 

1911  BOTTOMLET.     The  association  of  certain  endophytic  Cyanophyceae  and  nitro- 

gen-fixing bacteria.    Ept.  Brit.  Assoc.  Adv.  Sci.  (1911),  80,  786-787. 

BRIOSI,  G.  Untersuchungen  ueber  die  Assimilation  des  freien  atmospharis- 
chen  Stickstoffs  in  den  Pflanzen.  Centbl.  Agr.  Chem.  (1911),  40,  314. 

"FRED.  A  physiological  study  of  the  legume  bacteria.  Vir.  Agr.  Exp.  Sta. 
Ann.  Ept.  (1911),  145-174. 

GREIG-SMITH.  The  slime  or  gum  of  Ehizobium  leguminosarum.  Centbl.  f. 
Bakt.  2  Abt.  (1911),  30,  552-556. 

HARTWELL  AND  PEMBER.  The  gain  in  nitrogen  during  a  five  year  pot  experi- 
ment with  different  legumes.  E.  I.  Agr.  Exp.  Sta.  Bui.  .147  (1911). 

KELLERMAN.  The  relation  of  crown-gall  to  legume  inoculation.  U.  S.  Dept. 
Agr.  Bur.  Plant  Indus.  Cir.  76  (1911),  1-6. 

MARSHALL.     Microbiology.     (1911  edition),  273-283.     (Philadelphia) 

EITTER,  G.  Beitrage  zur  Stickstoffernahrung  der  Leguminosen  (Versuche  mit 
Lupinen  auf  schwerem  Boden).  Centbl.  Agr.  Chem.  (1911),  40,  767-773. 
Centbl.  f.  Bakt.  2  Abt.  (1911),  29,  650. 

SMITH,  E.  F.  Bacteria  in  relation  to  plant  diseases.  Carnegie  Inst.  Wash- 
ington Pub.  (1911),  2,  97-146. 

SMITH  AND  TOWNSEND.  Crown-gall  of  plants:  its  cause  and  remedy.  U.  S. 
Dept.  Agr.  Bur.  Plant  Indus.  Bui.  213  (1911). 

ZDRODOWSKI,  J.  DE.  Contribution  a  1 'etude  des  tubercules  radicaux  des  Le- 
gumineuses.  Eicherches  sur  1'onobrychis  Sativa  Fam.  (1911),  847  p. 
with  plates.  (Grenoble) 

1912  BREDEMANN.     Untersuchungen    ueber     das    Bakterien    Impfpriiparat    usw. 

Landw.  Jahrb.  (1912),  43,  669-694. 
DUGGAR  AND  PRUCHA.     The  behavior  of  Pseudomonas  radicicola  in  the  soU. 

Centbl.  f.  Bakt.  2  Abt.  (1912),  34,  67. 
EDWARDS.     Lebenfahigkeit  des  Ps.  radicicola  auf  Maltoseagar.     Centbl.  f. 

Bakt.  2  Abt.  (1912),  32,  199-200. 
GREIG-SMITH.     The  determination  of  rhizobia  in  the  soil.    Centbl.  f .  Bakt.  2 

Abt.  (1912),  34,  227-229. 
36KELLERMAN.     The  present  status  of  soil  inoculation.     Centbl.   f.  Bakt.   2 

Abt.   (1912),  34,  42-50. 
SEWERIN.     Die  Mobilisicrung  der  Phosphorsaure  des  Bodens  unter  dem  Ein- 

fluss  der  Lebenstatigkeit  der  Bakterien.    Centbl.  f.  Bakt.  2  Abt.  (1912.), 

32,  502. 

ZEEUW.  The  comparative  viability  of  seeds,  fungi,  and  bacteria,  when  sub- 
jected to  various  chemical  agents.  Centbl.  f.  Bakt.  2  Abt.  (1912),  31, 

5-23. 
37ZiPFEL.     Beitrage  zur  Morphologic  und  Biologic  der  Knollchenbakterien  der 

Leguminosen.     Centbl.  f.  Bakt.  2  Abt.   (1912),  32,  97-137. 

1913  EWART  AND  THOMSON.     On  the  cross  inoculation  of  the  root  tubercle  bac- 

teria upon  the  native  and  the  cultivated  Leguminosae.  Exp.  Sta.  Eec. 
(1913),  29,  733. 

FISCHER.  Die  Bakterienimpfung  zu  Legumino£en.  Gartenflora  (1912),  62, 
42-43.  Centbl.  f.  Bakt.  2  Abt.  (1915),  41,  286. 

HERKE.  Contribution  on  nitrogen  fixation  and  nutrition  of  Bacillus  radici- 
cola and  on  bacterial  tests  of  nitragin  and  azotogen.  Kiserlet  Kozlem. 
(1913),  16,  No.  3,  311-312.  Abst.  in  Exp.  Sta.  Eec.  (1913),  29,  733. 


178  BULLETIN  No.  202 

KELLERMAN  AND  LEONARD.    The  prevalence  of  Bacillus  radicicola  in  soil. 

Science  (1913),  N.  S.  38,  95-98. 
LOHNIS.     Vorlesungen  ueber  landwirtschaftliche  Bakteriolpgie   (1913),  161- 

170,  363-378.     (Berlin) 
MARSHALL.     The  utilization  of  atmospheric  nitrogen  by  natural  and  artificial 

means.    Exp.  Sta.  Eec.  (1913),  29,  821. 

1914  FEILITZEN    VON    u.   NYSTROM.     Neue  Impfsversuche   auf  Jung  farulichem 

Hochmoorboden   mit  verschiedenden  Leguminosen   Bakterien  Culturen. 

Jour.  f.  Landw.  (1914),  62,  285. 
MGARMAN  AND  DiDLAKE.     Six  different  species  of  nodule  bacteria.    Ky  Agr. 

Exp.  Sta.  Bui.  184  (1914). 
HEINZE.     Einige  weitere  Beitrage  zur  Kultur  der  Leguminosen  mit  besonderer 

Berucksichtigung   der    Stickstoffernahrun'g.     Centbl.   f.   Bakt.    2     Abt. 

(1914),  41,  285-286. 
*9KLIMMER  UNO  KRUGER.     Sind  die  beiden  verschiedenen  Leguminosen  ge- 

funden   Knollchenbakterien    artverschieden  ?     Centbl.    f.   Bakt.   2   Abt. 

(1914),  40,  256-265. 
KOVESSI.     The  assimilation  of  atmospheric  nitrogen  by  plant  hairs.     Bev. 

Gen.  Bot.  (1914),  25,  405-415;    and  26,  22-47,  106-128.     Exp.  Sta.  Eec. 

(1915),  32,  327. 
KRUGER.    Eelationships  among  nodule  bacteria  of  some  legumes.     Exp.  Sta. 

Bee.  (1915),  33,  823.    Ztschr.  Bot.  (1914),  6,  782-783. 
*°SiMON.     Ueber  die  Verwandtschaftsverhaltnisse   der  Leguminosen-Wurzel- 

bakterien.    Centbl.  f.  Bakt.  2  Abt.  (1914),  41,  470-479. 

1915  BARTHEL.    Culture  experiments  with  bacterial  inoculations  of  lupins  and 

alfalfa.    Exp.  Sta.  Eec.  (1915),  32,  433. 

GILTNER.     Nodule  formation.    Mich.  Ept.  State  Board  Agr.  (1915),  206-207. 
HARRISON.     Nitro-cultures  and  their  commercial  applications.     Trans.  Eoy. 

Soc.  Can.  (1915),  Ser.  3,  9,  219. 
KNO.     Influence  of  certain  salts  on  nodule  production  in  vetch.    Eead  at  9th 

meeting  Bot.  Soc.  of  Amer.  Abst.  in  Science  (1915),  N.  S.  41,  180. 
Kb'CK.     Die  Verwendung  von   Knollchenbakterien  zu   Leguminosen  Vienna 

Mitt.    Pflanzenschutz  Sta.     (1915),  4  pi.,  1  fig. 
41LiPMAN,  C.  B.,  AND  FOWLER.     Isolation  of  Bacillus  radicicola  from  soil. 

Science  (1915),  N.  S.  41,  256-259. 
42LiPMAN,  C.  B.     Isolation  of  B.  radicicola  from  soil.    Science  (1915),  N.  S. 

41,  725. 
OLARU.     Favorable  action  of  manganese  on  the  bacteria  of  leguminous  plants. 

Compt.  Eend.  Acad.  Sci.   (Paris)    (1915),  160,  280-283.     Abst.  in  Exp. 

Sta.  Eec.  (1915),  33,  820. 
43PRUCHA.     Physiological  studies  of  Bacillus  radicicola  of  Canada  field  pea. 

N.  Y.  (Cornell)  Agr.  Exp.  Sta.  (1915)  Memoir  5. 
WHITING.     A  biochemical  study  of  nitrogen  in  certain  legumes.     111.  Agr. 

Exp.  Sta.  Bui.  179  (1915). 
WILSON.     Physiological  studies  of  Bacillus  radicicola  of  soybean.    Eead  at 

9th  Meeting  Bot.  Soc.  of  Amer.    Abst.  in  Science  (1915),  N.  S.  41,  180. 

See  also  N.  Y.  (Cornell)  Agr.  Exp.  Sta.  (1917),  Bui.  386. 

1916  FRED  AND  GRAUL.     The  effect  of  soluble  nitrogenous  salts  on  nodule  forma- 

tion.    Jour.  Amer.  Soc.  Agron.   (1916),  8,  316. 
L0HNIS  AND  SMITH.     B.  radicicola.     Life  cycles   of  bacteria.     Jour.  Agr. 

Ees.  (1916),  6,  675. 
TEMPLE.     Studies  of  Bacillus  radicicola.     I,  Testing  commercial  cultures; 

II,  Soil  as  a  medium.    Georgia  Agr.  Exp.  Sta.  Bui.  120  (1916). 


1917]   POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES       179 

(b)       NON-LEGUME  BOOT  NODULES 

1860  SCHACHT.     Wurzelwucherungen.    Der  Baum  (1860),  172-174. 

1866  WOEONIN.     Ueber  die  bei  der  Schwarzerle    (Alnus  glutinosa)   imd  bei  der 

gewohnlichen  Gartenlupine    (Lnpinus  mutabilis)    auftretenden  Wurzel- 

anschwellungen.    Mem.  Acad.  Imp.  Sci.  (St.  Petersburg)   (1866),  Ser.  7, 

10,  No.  6,  1-13. 
1872  REINKE.     Parasitische   Anabaena   in   Wurzeln    der    Cycadeen.   Gott.  Nachr. 

(1872),  107. 
SCHENK.     Ueber  Cycas  Wurzeln  mit  Algencolonien.     Tagebl.  45  Versamml. 

Deut.  Naturf.  Aertze  Leipzig  (1872). 
1876  WARMING.     Wurzelknollchen  bei  den  Elaeagnaceen.     Just's  Bot.  Jahresber. 

(1876),  4,  439. 

1879  GRAVIS.     Observation  anatomiques  sur  les  excroissances  des  racines  de  1  'aune. 

Bui.  Soc.  Eoy.  Bot.  Belgique  (1879),  18,  1  Partie,  50-60. 
GRAVIS.     Le  Schinzia  alni  Woronine.    Mem.  Soc.  Eoy.  Bot.  Belgique  (1879), 
18,  50-60. 

1880  FRANK.     Wurzelanschwellungen   der  Erie.      Die   Krankheiten   der  Pflanzen 

(1880),  648.     (Breslau) 
GRAVIS.     Note  sur  les  excroissances  des  recines  de  1  'aune.  Compt  Eend.  Seance 

Mensuelle,  Jan.  10,  1880. 
WORONIN.     Nouvelles  recherehes  sur  le  genre  Schinzia.     Eevue  Mycologique 

(1880),  2,  69-70. 

1885  BRUNCHORST.     Ueber  die  Knb'llchen  an   den  Wurzlen  von  Alnus   und  den 

Elaeagnaceen.    Bot.  Centbl.  (1885),  24,  222. 
MOLLER.     Ueber  Plasmodiophora  alni.    Ber.    Deut.    Bot.    Gesell.     (1885),  3, 

102-105. 

WORONIN.     Bemerkung  zu  dem  Aufsatze  von  Herr  H.  Moller  ueber  Plam- 
odiophora  alni.    Ber.  Deut.  Bot.  Gesell.  (1885),  3,  177-178. 

1886  BRUNCHORST.     Ueber  einige  Wurzelanschwellungen,  besonders  diejenigen  von 

Alnus   und    den   Elaeagnaceen.     Unter.  Bot.  Inst.  Tubingen  (1886),  2, 

151-177.    Centbl.  f.  Agr.  Chem.  (1888),  17,  211. 
BRUNCHORST.      Ueber    einige    Wurzelanschwellungen    von    Alnus    und    der 

Elaeagnaceen.    Unters.  Bot.  Inst.  Tubingen  (1886).    Bot.  Centbl.  (1886), 

28,  109. 
SORAUER.     Wurzelanschwellungen  der  Erie.     Pflanzenkrankheiten   (1886),  2 

Aufl.,  1,  747. 

1887  FRANK.     Sind  die  Wurzelanschwellungen  der  Erlen  und  Elaeagnaceen  Pilz- 

gallen?    Ber.  Deut.  Bot.  Gesell.  (1887),  5,  50-57. 

1890  BEAL.     Tubercles  on  Ceanothus  americanus.    Bot.  Gaz.  (1890),  15,  232. 
MOLLER.     Beitrage   zur   Kenntniss   der   Frankia   subtilis   Brunchorst.      Ber. 

Deut.  Bot.  Gesell.  (1890),  8,  215-224.    Bot.  Centbl.  (1890),  45,  60-61. 

1891  ATKINSON.     Tubercles  of  Ceanothus.    Bot.  Gaz.  (1891),  16,  262. 

FRANK,  B.  Ueber  die  auf  Verdauung  von  Pilzen  abzielende  Symbiose  der 
mit  endotrophen  Mykorrhizen  begabten  Pflanzen,  sowie  der  Leguminosen 
und  Erlen.  Ber.  Deut.  Bot.  Gesell.  (1891),  9,  244-253. 

1892  "ATKINSON.     The  genus  Frankia  in  the  United  States.    Bui.  Torrey  Bot.  Club 

(1892),  19,  171-177. 

NOBBE,  SCHMID,  HiLTNER,  UND  HOTTER.  Ueber  die  physiologische  Bedeutung 
den  Wurzelknollchen  von  Elaeagnus  angustifolius.  Landw.  Vers.  Stat. 
(1892),  41,  138-140. 

1894  SCHNEIDER.     Mutualistic  symbiosis  of  algae  and  bacteria  with  Cycas  revoluta. 

Bot.  Gaz.  (1894),  19,  25-32. 

1895  TUBEUF,  VON.     Mykodomatien  der  Erlen,  Elaeagnaceen,  und  Myricaceen,  ver- 

anlasst   durch  Frankia  Arten.     Pflanzenkrankheiten  durch  kryptogame 
Parasiten  verursacht   (1895),  1-117.      (Berlin) 


180  BULLETIN   No.  202  [July, 

1896  43HiLTNER.  Ueber  die  Bedeutung  der  Wurzelknollchen  von  Alnus  glutinosa 
fur  die  Stickstoffernahrung  dicser  Pflanzc.  Landw.  Vers.  Stat.  (1896), 
46,  153-161. 

1898  HILTNER.     The  root  tubercles  of  alders  and  the  Elaeagnaccae.    Exp.  Sta.  Eec. 

(1898),  10,  825.    Forstl.  Natur.  Ztschr.  (1898),  7,  415-423. 
48NOBBE  UND  HILTNER.     Die  endotrophe  Mykorrhiza  von  Podocarpus  und  ihre 
physiologische  Bedeutung.    Landw.  Vers.  Stat.  (1898),  51,  241-245. 

1899  HILTNER.     Ueber  die  Assimilation   des   freien    atmospharischen  Stickstoffs 

durch  in  oberirdischen  Pflanzenteilen  lebende  Mycelien.  Centbl.  f.  Bakt. 
2  Abt.  (1899),  5,  831-837. 

1900  WOODHEAD.     On  the  structure  of  root  nodules  of  Alnus  glutinosa.    Kpt.  Brit. 

Assoe.  Adv.  Sci.  (1900),  70,  931. 

1901  47LiFE.     The  tuber-like  rootlets  of  Cycas  revoluta.     Bot.  Gaz.    (1901),  31, 

265-271. 

PAMPALONI.  II  Nostoc  punctiforme  nei  suci  rapparti  coi  tubercoli  radicoli 
delle  Cicadee.  Nuovo  Giorn.  Ital.  (1901),  N.  S.  8,  626-632.  Just's  Bot. 
Jahresber.  (1901),  29,  Part  1,  307. 

1902  SHIBATA.     Cytologische  Studien  ueber  die  endotrophen  Mykorrhiza.     Jahrb. 

Wiss.  Bot.  (1902),  37,  643-684. 

1903  HARSHBARGER.     The  form  and  structure  of  the  mycodomatia  of  Myrica  ceri- 

fera.     Proc.  Acad.  Nat.  Sci.  Phila.  (1903),  55,  352-361. 

HILTNER.  Beitrage  zur  Mykorrihizafrage.  Ueber  die  biologische  und  physi- 
ologische  Bedeutung  der  endotrophen  Mykorrhiza.  Naturw.  Ztschr.  Land. 
Forstw.  (1903),  1,  9.  Centbl.  f.  Bakt.  2  Abt.  (1903),  10,  479. 

1904  BJORKENHEIM.     Keitrage  zur  Kenntniss  des  Pilzes  in  den  Wurzelanschwellun- 

gen  von  Alnus  incana.    Ztschr.  Pflanzenkrankh.  (1904),  14,  128-134. 

1906  BOTTOMLEY.     The  cross-inoculation   of   Leguminosae   and   other  root-nodule 

bearing  plants.    Ept.  Brit.  Assoc.  Adv.  Sci.  (1906),  76,  752-753. 

1907  BOTTOMLEY.     The  structure  of  root  tubercles  in  leguminous  and  other  plants. 

Ept.  Brit.  Assoc.  Adv.  Sci.  (1907),  77,  693. 

1908  ZACH.     Ueber  den  in  den  Wurzelknollchen  von  Elaeagnus  angustifolia  und 

Alnus  glutinosa  lebendeu  Fadenpilz.  Situngsb.  K.  Akad.  Wiss.  Wien. 
Mat.  Nat.  Klasse  (1908),  117,  973-983.  Centbl.  Agr.  Chem.  (1910),  39, 
622. 

1909  4SBOTTOMLEY.     Some  effects  of  nitrogen-fixing  bacteria  on  the  growth  of  non- 

leguminous  plants.    Proc.  Boy.  Soc.  London  (1909),  81,  B,  287. 
BOTTOMLEY.     Nitrogen  fixing  bacteria  and  non-leguminous  plants.   Nature 

(1909),  82,  218. 
HALL.     Nitrogen  fixing  tacteria  and  non-leguminous  plants.     Nature  (1909), 

82,  218-219. 
MAIRE  ET  TISON.     La  cytologie  des  Plasmodiophoracees  et  la  classe  des  Phy- 

tomyxinae.    Ann.  Mycol.  (1909),  7,  226-253. 
PEKLO.     Beitrage   zur   Losung   des   Mykorrhizaproblems.      Ber.   Deut.   Bot. 

Gesell.  (1909),  27,  239-247. 

TISON.     Sur  le  Saxegothaea.    Lindl.  Mem.  Soc.  Linn.  Normandie  (1909),  23. 
WOLPERT.     Die  Mykorrhizen  von  Alnus.     Flora  (1909),  100,  60-67. 

1910  49ARZBERGER.     The  fungous  root-tubercles  of  Ceanothus  americanus,  Elaeag- 

nus Argentea,  and  Myrica  cerifera.  Mo.  Bot.  Gardens  21st  Ann.  Ept. 
(Dec.,  1910),  60-103.  • 

BOTTOMLEY.  The  fixation  of  nitrogen  by  free  living  soil  bacteria.  Ept.  Brit. 
Assoc.  Adv.  Sci.  (1910),  80,  581. 

BOTTOMLEY.  The  association  of  certain  endophytic  Cyanophyceae  and  nitro- 
gen-fixing bacteria.  Ept.  Brit.  Assoc.  Adv.  Sci.  (1910),  80,  786. 

BOTTOMLEY.  Nitrogen-fixing  bacteria  and  non-leguminous  plants.  Nature 
(1910),  83,  96. 


1017}   POSSIBLE  SYMBIOSIS  BETWEEN  LEGUME  BACTERIA  AND  NON-LEGUMES       181 

KELLERMAN.     Nitrogen  gathering  plants.    U.  S.  Dept.  Agr.  Yearbook  (1910), 

213-218. 
PEKLO.     Die  Pflanzenlichen  Aktinomykosen.    Gentbl.  f.  Bakt.  2  Abt.  (1910), 

27,  451-579. 
ZACH.     Studio  ueber  Phagocytose  in  den  Wurzelknollchen  der  Cycadeen.    Bot. 

Ztschr.  (1910),  60,  49-55. 

1911  BOTTOMLEY.     The  structure  and  function  of  the  root  nodules  of  Myrica  gale. 

Bpt.  Brit.  Assoc,  Adv.  Sci.  (1911),  81,  584. 
BOTTOMLEY.     The  fixation  of  nitrogen  by  free  living  soil  bacteria.    Ept.  Brit. 

Assoc.  Adv.  Sci.  (1911),  81,  GOT. 
BOTTOMLEY.     The  structure  and  physiological  significance  of  the  root  nodules 

of  Myrica  gale.    Proc.  Roy.  Soc.  London  (1911),  84,  B,  213-216. 
WSPRATT.     Some  observations  on  the  life-history  of  Anabaena  cycadeae.    Ann. 

Bot.  (1911),  25,  369-381.  • 

1912  "BOTTOMLEY.     The  root  nodules  of  Myrica   gale.     Ann.   Bot.    (1912),  26, 

111-116. 

BOTTOMLEY.  The  root  nodules  of  Podocarpeae.  Ept.  Brit.  Assoc.  Adv.  Sci. 
(1912),  82,  679. 

B2SPRATT.  The  Morphology  of  the  root  tubercles  of  Alnus  and  Elaeagnus, 
and  the  polymorphism  of  the  organism  causing  their  formation.  Ann. 
Bot.  (1912),  26,  119-128. 

53SPRATT.  The  formation  and  physiological  significance  of  root  nodules  in  the 
Podocarpineae.  Ann.  Bot.  (1912),  26,  801-814. 

1915  "BOTTOMLEY.     The  root  nodules  of  Ceanothus  americanus.    Ann.  Bot.  (1915), 
29,  605-611. 

65SPRATT.  The  root  nodules  of  the  Cycadaceae.  Ann.  Bot.  (1915),  29,  619- 
621. 


UNIVERSITY  OF  ILLINOIS-URBANA 


